1 The effect of L-NG-nitro arginine (L-NOARG) was compared with that of L-NG-monomethyl arginine (L-NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2 L-NOARG (1.5-100Mm) and L-NMMA (3-100Mm) produced concentration-related contraction of the rabbit aorta precontracted with phenylephrine (700-900nM). Similarly, L-NOARG (10-2001pM) and L-NMMA (30-100 gM) elevated perfusion pressure of the noradrenaline (NA, 0.6-2.5mM)-preconstricted rat mesentery preparation. 6 These results identify L-NOARG as a potent, L-arginine reversible inhibitor of endothelium-dependent vasodilatation. The available data suggests that L-NOARG, like L-NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

Contraction of smooth muscle is initiated, and to a lesser extent maintained, by a rise in the concentration of free calcium in the cell cytoplasm ([Ca 2+ ] i ). This activator calcium can originate from two intimately linked sources ± the extracellular space and intracellular stores, most notably the sarcoplasmic reticulum. Smooth muscle contraction activated by excitatory neurotransmitters or hormones usually involves a combination of calcium release and calcium entry. The latter occurs through a variety of calcium permeable ion channels in the sarcolemma membrane. The bestcharacterized calcium entry pathway utilizes voltage-operated calcium channels (VOCCs). However, also present are several types of calcium-permeable channels which are non-voltage-gated, including the so-called receptor-operated calcium channels (ROCCs), activated by agonists acting on a range of G-protein-coupled receptors, and store-operated calcium channels (SOCCs), activated by depletion of the calcium stores within the sarcoplasmic reticulum. In this article we will review the electrophysiological, functional and pharmacological properties of ROCCs and SOCCs in smooth muscle and highlight emerging evidence that suggests that the two channel types may be closely related, being formed from proteins of the Transient Receptor Potential Channel (TRPC) family.

The effects of L-NG-nitro arginine (L-NOARG) on a-chymotrypsin-resistant, non-adrenergic, noncholinergic (NANC) relaxations of guinea-pig tracheal smooth muscle have been examined. L-NOARG (1-100pM), but not D-NOARG (100pM), inhibited the NANC relaxations in a concentration-related manner. The effects of L-NOARG were partially reversed by L-arginine but not D-arginine. L-NOARG was without effect on acetylcholine-induced contractile responses of the trachea or on relaxations produced by vasoactive intestinal peptide, sodium nitroprusside or isoprenaline. These results suggest that an endogenous nitrate may contribute to NANC relaxations of tracheal smooth muscle.Introduction In many species, including man and guinea-pig, electrically induced relaxations of tracheal smooth muscle have a non-adrenergic, non-cholinergic (NANC) component (Barnes, 1986). The identity of the neurotransmitter which mediates this effect is unknown and may vary with the species studied. In the guinea-pig, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are strong candidates for at least part of this NANC relaxant response. Ellis &

1 By use of the whole-cell configuration of the patch-clamp technique, membrane currents induced by cyclopiazonic acid (CPA; an inhibitor of the sarcoplasmic reticulum (SR) calcium-ATPase) were investigated in single smooth muscle cells freshly dispersed from the mouse anococcygeus. Voltagedependent calcium currents were blocked with extracellular nifedipine and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. 2 At a holding potential of -40 mV, CPA (10 tiM) activated an inward current that consisted of two distinct components. The first was an initial transient current with an amplitude of 19.6 + 1.9 pA while the second was sustained and had an amplitude of 3.5 + 0.3 pA. 3 The current-voltage (I-V) relationship for the transient current showed marked outward rectification. The current had a reversal potential of 9.1 + 1.1 mV which was shifted to 29.0 + 4.2 mV when the extracellular chloride concentration was lowered from 148.4 to 58.4 mM. The sustained current had a near-linear I-V relationship and a reversal potential of 31.0+2.7 mV. Removal of extracellular calcium had no effect on the transient current, but shifted the reversal potential of the sustained current to 18.2+ 5.7 mV. 3 The initial transient current was abolished in cells bathed in extracellular solutions containing the chloride channel blockers, 4,4' diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS; 1 mM) or anthracene-9-carboxylic acid (A-9-C; 1 mM), and was absent in cells containing the calcium buffers EGTA (1 to 5 mM) or BAPTA (10 mM). The second sustained current was unaffected by either the chloride channel blockers or the intracellular calcium buffers. 4 Treatment of the cells with caffeine (10 mM) produced similar inward currents to those produced by CPA. In the presence of caffeine, CPA (10 gM) induced no further inward current. 5 In organ bath studies, CPA (10 gM)-induced contractions of the mouse anococcygeus were inhibited by cadmium and nickel (both 50-400 uM) and the general calcium entry blocker, SKF 96365 (10 gM); lanthanum and gadolinium had no effect at concentrations up to 400 gM. The pharmacology of the CPA-induced non-selective cation current mirrored that of the CPA-induced whole muscle contraction being reversed by cadmium (100 tM) and SKF 96365 (10 uM), but unaffected by lanthanum (400 gM).The initial chloride conductance was unaffected by cadmium, SKF 96365 or lanthanum. 6 It is concluded that CPA activates a transient calcium-dependent chloride current as a consequence of calcium release from intracellular stores; this current would result in depolarization and opening of voltage-operated calcium channels, which mediate the nifedipine-sensitive component of muscle contraction. In addition, as a result of emptying the SR, CPA activates a non-selective cation conductance which may underlie the nifedipine-insensitive calcium entry process utilised during sustained contraction.

6LX1 The aim of this study was to determine whether the fish neuropeptide, Gillichthys urotensin II (GUII), possesses significant biological activity on rat aortic strips. 2 On intact strips, pre-contracted by noradrenaline (100 nM), low concentrations (0.1-0.5 nM) of GUII produced relaxations, while higher concentrations (1-10 nM) caused further contraction. On strips rubbed to remove endothelial cells, relaxations were absent but contractile responses to higher concentrations of GUII remained. 3 GUII (0.2-10nM) produced dose-related contractions of quiescent, intact aortic strips. These contractions consisted of two components, tonic and phasic, and were potentiated in rubbed strips and in the presence of the antioxidant drug hydroquinone (1O iM).4 Mepacrine (40 jM) and p-bromophenacyl bromide (50 gM) completely abolished contractions to GUII, but indomethacin (10 jM) and nordihydro-guaiaretic acid (1I0 M) were without effect. 5 The phasic, but not the tonic, component of the contractile response was inhibited by nitrendipine (200 nM), and was absent in bathing medium from which Ca2' had been omitted. Addition of EGTA (2 mM) to Ca2+-free bathing medium abolished the residual tonic component. 6 GUII-induced contractions were completely abolished by the calmodulin antagonists trifluoperazine (50 pM) and W-7 (30 gM).7 It is concluded that GUII, previously considered devoid of significant activity on mammalian tissues, produces potent endothelium-dependent relaxations and endothelium-independent contractions of rat aorta, and possible mechanisms underlying each response are discussed.

1The effects of L-N0-monomethyl arginine (L-NMMA) and L-N0-nitro arginine (L-NOARG) on nonadrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus were investigated. 2 L-NMMA (10-200pM) produced a concentration-related inhibition of the NANC response; the inhibitory effect of 5OPM L-NMMA was completely reversed by L-arginine but not D-arginine (both 100pM).3 L-NOARG (1-50,pM) also produced a concentration-related inhibition of the NANC response and was some 30-50 times more potent than L-NMMA; again, the effects of 10pM L-NOARG were reversed by 100pM L-, but not D-, arginine. By itself 100pM L-arginine did not relax the tissue, but did cause a slight potentiation of the NANC response.4 Sodium nitroprusside (0.01-lOuM), hydroxylamine (0.1-100 pM), sodium azide (1-100pM) and nitric oxide (3-120pM) all relaxed carbachol-induced tone; relaxations to submaximal concentrations of these nitrovasodilators were unaffected by either 50M L-NMMA or 10pM L-NOARG. 5 L-NOARG 10pM did not inhibit, but rather potentiated, contractions of the mouse anococcygeus due to stimulation of its sympathetic nerves. 6 The inhibitory effects of 10pM L-NOARG on NANC relaxations were reversed by L-arginine (by 131%), L-citrulline (by 75%), L-arginine methyl ester (by 46%) and L-homoarginine (by 22%), but were unaffected by a variety of other amino acids and their derivatives (all at 100 M). 7 The results provide strong evidence that NANC relaxations of the mouse anococcygeus are mediated by an endogenous nitrate material, probably derived from L-arginine, and confirm that L-NOARG provides a very useful and potent drug for the investigation of endogenous nitrate function.

1 The influence of hydroquinone on relaxations induced by nitric oxide (NO), nitrovasodilator drugs, and non-adrenergic, non-cholinergic (NANC) field stimulation has been investigated in three tissues in which endogenous nitrates have been implicated in the NANC response; the mechanism of action of hydroquinone was also studied. 2 In mouse anococcygeus, hydroquinone (10-1OOpM) produced a concentration-dependent inhibition of relaxations induced by 1S5UM NO. Hydroquinone, 100pMm, which reduced responses to NO by 85%, had no effect on relaxations induced by NANC field stimulation (1OHz; 20s trains), hydroxylamine (1OpM), sodium nitroprusside (1 gM) or sodium azide (20juM).3 In guinea-pig trachea, 100pM hydroquinone reduced relaxations to 150pM NO by 75%, but had no effect on those to NANC stimulation (1OHz; 30s trains) or sodium azide (5 pM).4 In rat gastric fundus, 100pUM hydroquinone reduced relaxations to 1 ,M NO by 85%, but had no effect on those to NANC stimulation (0.5 Hz; 15 s trains) or sodium azide (2pM). 5 Superoxide dismutase (SOD; 50uml-1) had no effect on relaxations of the mouse anococcygeus in response to 15,UM NO or 10Hz NANC stimulation. Further, the inhibition of responses to NO by hydroquinone was unaffected in the presence of SOD. 6 Hydroquinone (10-1OOuM) failed to generate superoxide anions, as detected by a chemiluminescent assay. However, 100piM hydroquinone, like SOD (50uml-1), produced almost complete inhibition of superoxide anion chemiluminescence induced by xanthine (500pgM): xanthine oxidase (0.07 u ml-1). 7 It is concluded that, in our system, hydroquinone inhibits NO by acting as a free radical scavenger rather than by generating superoxide anions. The ability of hydroquinone to block relaxations to NO, but not NANC stimulation, may suggest that the endogenous nitrate substance released by these NANC nerves may not be free NO, but may be an NO-containing, or NO-generating, molecule.

The potential protective effect of several antioxidants [Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbate, reduced glutathione (GSH), and cx-tocopherol (a-TOC)] on relaxations of the mouse anococcygeus muscle to nitric oxide (NO; 15 giM) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated.2 The superoxide anion generating drug duroquinone (100 gIM) reduced relaxations to exogenous NO by 54+6%; this inhibition was partially reversed by Cu/Zn SOD (250 u ml-'), and by ascorbate (500 gM). Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 gM) also reduced relaxations to nitrergic field stimulation (by 53 + 6%) and this effect was again reversed by Cu/Zn SOD and by ascorbate. Neither GSH (500 gM) nor (X-TOC (400 gM) afforded any protection against duroquinone.3 Xanthine (20 mu ml-1):xanthine oxidase (100 Mm) inhibited NO-induced relaxations by 73 + 14%, but had no effect on those to nitrergic field stimulation, even after DETCA treatment. The inhibition of exogenous NO was reduced by Cu/Zn SOD (250 u ml-1) and ascorbate (400 gM), but was unaffected by GSH or oc-TOC (both 400 gM). 4 Hydroquinone (100 gM) also inhibited relaxations to NO (by 52 + 10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5-100 Mm) and ascorbate (100-400 gM), although Cu/Zn SOD and a-TOC were ineffective. 5 2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 50 gM) inhibited NO-induced relaxations by 50+4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2-200 gM) and a-TOC (10-200 gM), but not by Cu/Zn SOD or GSH. 6 Hydroxocobalamin (5 -1000 gM) inhibited, equally, relaxations to both NO (-logIC40 3.14 + 0.33) and nitrergic stimulation (-logIC40 3.17+0.22). 7 Thus, a number of physiological antioxidants protected NO from superoxide anions, and from direct NO-scavengers. The possibility that the presence of these antioxidants within nitrergically-innervated tissues might explain the lack of effect of the NO inhibitors on nerve-induced relaxation, without the need to invoke a transmitter other than free radical NO, is discussed.