Mouse Anococcygeus Research Articles

Receptor‐independent activation of Rho‐kinase‐mediated calcium sensitisation in smooth muscle

1. The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2. The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC(50) values against CCh and Tg of 8.4+/-3.3 (n=6) and 6.1+/-2.1 (n=7) micro M, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69+/-7% (n=4) inhibition using 10 micro M Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A. 3. In beta-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 micro M) and GTP (100 micro M). These responses to CCh and Tg were inhibited by Y27632 (10 micro M). 4. Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5. These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.

Calyculin-A inhibits nitrergic relaxations of the mouse anococcygeus

The aim was to determine whether blockade of store-operated Ca 2+ entry, or inhibition of Ca 2+ sensitisation, is the predominant mechanism by which neuronally released nitric oxide mediates relaxation of the mouse anococcygeus. Nitrergic relaxations to field stimulation (10 Hz, 10 s trains) were unaffected by the sarcoplasmic reticulum Ca 2+ ATPase blocking agent thapsigargin (100 nM), known to prevent nitric-oxide-induced inhibition of store-operated Ca 2+ entry. Conversely, the myosin phosphatase inhibitor calyculin-A (1 μM) caused almost complete abolition of nitrergic relaxations. The results provide evidence that inhibition of Ca 2+ sensitisation is the major cellular mechanism underlying nitrergic relaxation of the mouse anococcygeus.

Selective inhibition of thapsigargin-induced contraction and capacitative calcium entry in mouse anococcygeus by trifluoromethylphenylimidazole (TRIM).

This study examined the effects of trifluoromethylphenylimidazole (TRIM) on tone, and calcium entry, in mouse anococcygeus stimulated by either thapsigargin (Tg; 100 nM) which activates capacitative calcium entry (CCE), or high K (60 mM) which activates voltage-operated calcium channels. TRIM (1 - 333 microM) produced concentration-related relaxation of Tg-induced tone (EC(50), 42 microM) but was much less effective against high K. In single smooth muscle cells loaded with FURA-2, TRIM reduced the increase in fluorescence ratio produced by Tg but had no effect on that produced by high K. The relaxations of Tg-induced tone, and reduction in fluorescence ratio, were obtained in the presence of L-N(G)-nitroarginine and were thus independent of nitric oxide synthase inhibition; further, TRIM had no discernible effect on nitrergic responses. TRIM provides a novel drug for the selective inhibition of CCE and a template for the development of more potent inhibitors.

Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway.

1. The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. 2. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 microM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 microM). 3. In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. 4. In beta-escin skinned preparations, NO had no effect on tone induced by calcium (1 microM in the presence of 100 microM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. 5. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation.

Thapsigargin-induced tone and capacitative calcium influx in mouse anococcygeus smooth muscle cells.

The sarcoplasmic reticulum Ca-ATPase inhibitor thapsigargin (Tg; 0.4-100 nM) produced concentration-related, strong and sustained contractions of the mouse-isolated anococcygeus muscle; these contractions were dependent on extracellular calcium but were only partially reduced (by about 50%) in the presence of verapamil (10 and 100 microM). The verapamil-resistant component of the Tg-induced contraction was relaxed by the general calcium entry blockers SKF96365 (0.4-40 microM) and cadmium (50-300 microM), and by the tyrosine kinase inhibitor genistein (10-180 microM). In single smooth muscle cells loaded with Fura-2, addition of Tg (100 nM) to calcium-free medium produced a small, transient increase in fluorescence; subsequent addition of calcium (2.5 mM) produced a larger and sustained increase which was abolished on return to calcium-free conditions, but was only partially reduced by verapamil (10 microM; by about 30%). Manganese quenching of Fura-2 was enhanced in cells treated with Tg. The verapamil-resistant calcium influx was reduced by SKF96365 (20 microM) and to a lesser extent by genistein (40 microM); cadmium (200 microM) produced an initial decrease in fluorescence followed by a marked increase. These results demonstrate that, in the mouse anococcygeus, Tg can cause sustained contractions and elevations of calcium influx in the presence of verapamil; the time-course, calcium dependence and, although to a lesser extent, pharmacology of these effects generally support the proposal that excitation-contraction coupling in this tonic smooth muscle involves sustained capacitative calcium influx.

Correlation between store-operated cation current and capacitative Ca 2+ influx in smooth muscle cells from mouse anococcygeus

In mouse anococcygeus cells, simultaneous measurements of membrane currents and changes in intracellular Ca 2+ were obtained using “perforated-patch” whole-cell recordings and Fura-2 microfluorimetry. Carbachol (50 μM) and cyclopiazonic acid (10 μM) produced a biphasic inward current; a transient Ca 2+-dependent chloride current ( I ClCa), followed by a smaller, sustained current ( I DOC). This response was mirrored by a biphasic increase in the intracellular Ca 2+ concentration. SKF96365 (1-{β-[3-(4-methoxyphenyl) propoxyl]-4-methoxyphenethyl}-1 H-imidazole; 10 μM) and Cd 2+ (100 μM) inhibited both I DOC and the sustained increase in intracellular Ca 2+; La 3+ (400 μM) inhibited neither response. The results confirm that the non-selective cation current I DOC underlies capacitative Ca 2+ influx supporting sustained contractions in this tonic smooth muscle.

Characterisation of nitrergic transmission in the isolated anococcygeus muscle of the female mouse

Field stimulation of anococcygeus muscles from female mice produced frequency-dependent relaxations of carbachol-induced tone, which were independent of the oestrus cycle but were abolished by the nitric oxide synthase (NOS) inhibitor l- N G-nitroarginine ( l-NOARG; 100 μM) and the soluble guanylyl cyclase inhibitor 1 H-[1,2,4]oxodiazolo[4,3- a]quinoxalin-1-one (ODQ; 5 μM); l-NOARG inhibition was reversed by l-, but not d-arginine. The selective phosphodiesterase V inhibitor zaprinast (1–130 μM) directly relaxed tone and enhanced both the amplitude and duration of field stimulation-induced relaxations; the effect on amplitude was greater at lower frequencies of stimulation, while increased duration dominated at higher frequencies. The duration, but not the amplitude, of relaxations to exogenous nitric oxide (NO; 15 μM) was also increased by zaprinast. The mouse anococcygeus provides a useful model for pharmacological investigation of nitrergic neurotransmission in female urogenital smooth muscle.

No evidence for a significant non-nitrergic, hyperpolarising factor contribution to field stimulation-induced relaxation of the mouse anococcygeus.

1. The aim of the study was to determine whether a nerve-derived hyperpolarizing factor (NDHF) might contribute to non-adrenergic, non-cholinergic (NANC) relaxations of the mouse anococcygeus when low concentrations of contractile agent are used to raise tone and low frequencies of field stimulation applied; such a non-nitrergic NDHF has been proposed to contribute to NANC relaxations of the rat anococcygeus and guinea-pig taenia coli. 2. Phenylephrine (0.1-100 microM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 microM phenylephrine (EC26) was used to raise tone in subsequent experiments. 3. Field stimulation (0.5, 1.0 and 5.0 Hz) produced frequency-dependent relaxations of phenylephrine-induced tone. In the presence of the nitric oxide synthase inhibitor L-NG-nitro-arginine (L-NOARG; 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 microM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01-5 microM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 microM). 4. Nifedipine (1 microM) reduced the contractile response to 0.2 microM phenylephrine by 38%; however, it had no effect on NANC relaxations. 5. It is concluded that NANC relaxations of the mouse anococcygeus are purely nitrergic and that there is no significant contribution from a putative NDHF.

Depletion of either ryanodine- or IP3-sensitive calcium stores activates capacitative calcium entry in mouse anococcygeus smooth muscle cells.

The properties of the calcium stores coupled to a depletion-operated cation current (IDOC) proposed to underlie capacitative calcium entry were studied in single smooth muscle cells isolated from the mouse anococcygeus using the whole-cell patch-clamp technique. Both caffeine (10 mM) and carbachol (50 microM) activated an initial, large ( approximately 200 pA), transient, calcium-dependent chloride current (IClCa) followed by a smaller ( approximately 10 pA) sustained, non-selective cation current (IDOC). Intracellular application of heparin (5 mg ml-1) abolished the response to carbachol but potentiated that to caffeine. Ryanodine (3 microM) activated IDOC but not IClCa; ryanodine (30 microM) failed to produce any response. Both concentrations of ryanodine abolished the response to caffeine and prevented activation of IClCa by carbachol. In the presence of 30 microM, but not 3 microM, ryanodine, carbachol was able to activate IDOC. Cyclopiazonic acid (CPA; 10 microM) abolished the response to carbachol; however, caffeine was still able to activate IClCa. In whole-muscle tension recordings, ryanodine at both 3 and 30 microM produced contractions of the tissue but only that in response to the lower concentration was maintained. Thus, depletion of either inositol 1,4, 5-trisphosphate-(IP3-) sensitive or ryanodine-sensitive calcium stores is able to activate IDOC, and, by extension, capacitative calcium entry in this tissue.

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus.

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

The rat anococcygeus muscle and its response to nerve stimulation and to some drugs

The rat anococcygeus muscle and its response to nerve stimulation and to some drugs

Mouse anococcygeus muscle: sexual dimorphism and responsiveness to sex hormones.

The anococcygeus muscle (AcM) is one of a pair of thin sheets of smooth muscle inserting on the rectum, having a tendinous origin largely on sacral vertebrae. The cross-sectional area of AcM in the juxtarectal region in 90-day-old male mice was significantly larger than that in females of three strains: BALB/cCrgl, ICR/Jcl and C57BL/Tw. The AcM area in female mice showed strain differences: BALB/c > ICR > C57BL. Five daily injections of testosterone into newborn ICR mice from the day of birth significantly increased the areas of AcM in both sexes at 30 days of age, but five daily injections of oestradiol-17 beta (OE) decreased them. The AcM area in 60-day-old ICR male mice castrated at 30 days of age was significantly smaller than in intact males, and that in ovariectomized females was significantly larger than in intact females. In both sexes, implantation of a testosterone pellet (12 mg) into gonadectomized mice on the day of gonadectomy stimulated the growth of AcM, and implantation of an OE pellet (12 mg) inhibited the growth of AcM. The AcM in both ICR and C57BL strains showed positive androgen receptor and oestrogen receptor immunostaining at 15 days. Female ICR mice exposed neonatally to diethylstilboestrol (DES) had significantly larger AcM than controls; ovariectomy at 30 days of age did not change the AcM area in 60-day-old DES-exposed mice. However, male mice exposed neonatally to DES had significantly smaller AcM than controls; castration at 30 days of age nullified this inhibition. These results suggest that both androgen and oestrogen play an important role in sexual dimorphism of the mouse AcM. Neonatal exposure to DES (but not to oestradiol) had an irreversible stimulatory effect on the AcM area in female mice.

Antioxidant protection of NO-induced relaxations of the mouse anococcygeus against inhibition by superoxide anions, hydroquinone and carboxy-PTIO.

1. The potential protective effect of several antioxidants [Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbate, reduced glutathione (GSH), and alpha-tocopherol (alpha-TOC)] on relaxations of the mouse anococcygeus muscle to nitric oxide (NO; 15 microM) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated. 2. The superoxide anion generating drug duroquinone (100 microM) reduced relaxations to exogenous NO by 54 +/- 6%; this inhibition was partially reversed by Cu/Zn SOD (250 u ml-1), and by ascorbate (500 microM). Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 microM) 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 microM) nor alpha-TOC (400 microM) afforded any protection against duroquinone. 3. Xanthine (20 mu ml-1); xanthine oxidase (100 microM) 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 microM), but was unaffected by GSH or alpha-TOC (both 400 microM). 4. Hydroquinone (100 microM) also inhibited relaxations to NO (by 52 +/- 10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5-100 microM) and ascorbate (100-400 microM), although Cu/Zn SOD and alpha-TOC were ineffective. 5. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 50 microM) inhibited NO-induced relaxations by 50 +/- 4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2-200 microM) and alpha-TOC (10-200 microM), but not by Cu/Zn SOD or GSH. 6. Hydroxocobalamin (5-100 microM) 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.

Inhibition by sodium nitroprusside of a calcium store depletion-activated non-selective cation current in smooth muscle cells of the mouse anococcygeus.

1. The effects of sodium nitroprusside (SNP) on the non-selective cation current activated in response to intracellular calcium store depletion were studied using the whole-cell patch-clamp technique in single smooth muscle cells isolated from the mouse anococcygeus. Voltage-dependent calcium currents were blocked with extracellular nifedipine, and caesium and tetraethylammonium chloride were used to block voltage-dependent potassium currents. Calcium stores were depleted with caffeine (10 mM), carbachol (50 microM) or cyclopiazonic acid (CPA 10 microM; an inhibitor of the sarcoplasmic reticulum [SR] calcium-ATPase). 2. At a holding potential of -40 mV, both CPA and caffeine activated inward currents which consisted of two clearly distinguishable components; an initial transient current followed by a smaller sustained current. In the case of CPA, the amplitudes of the transient and sustained components were 19.7 +/- 2.1 pA and 3.5 +/- 0.3 pA respectively, whilst the equivalent values for caffeine were 188 +/- 21 and 4.8 +/- 0.3 pA. As described previously, the transient current results from activation of a calcium-dependent chloride conductance whilst the sustained current is a non-selective cation current, activated following intracellular calcium store depletion. 3. The muscarinic receptor agonist, carbachol, also activated a transient followed by a sustained current with amplitudes of 238 +/- 55 and 4.7 +/- 0.5 pA respectively. Superimposed on the sustained current were regular, oscillations of calcium-activated chloride current. 4. Both the transient and the sustained currents activated by CPA were absent in cells pretreated with SNP (10 microM). Application of SNP to a cell following activation of the sustained current by CPA inhibited the current by 88.6 +/- 3.8%. SNP (10 microM) did not inhibit the transient current activated by caffeine but abolished the sustained current. 5. SNP (10 microM) had no effect on the initial transient current activated by carbachol (50 microM). However, it did inhibit the oscillations in the inward current. In recordings from cells bathed in extracellular solution containing the chloride channel blocker, anthracene-9-carboxylic acid (A-9-C; 1 mM), carbachol activated only a sustained current. This current was inhibited by 88.1 +/- 6.5% by a concomitant application of SNP (10 microM) and was absent in cells pretreated with the nitrovasodilator. 6. The effects of SNP on the currents activated by caffeine (10 mM) were mimicked by 8-bromo-cyclic GMP (200 microM); thus the nucleotide had no effect on the transient current activated by caffeine but abolished the sustained current. The effects of SNP, but not those of 8-bromo-cyclic GMP, were inhibited by the nitric oxide-sensitive guanylyl cyclase inhibitor, 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ; 1 microM). ODQ alone produced a significant increase in the size of the sustained current activated by caffeine (7.8 +/- 0.7 pA). 7. These findings suggest that SNP activates guanylyl cyclase to inhibit the non-selective cation current activated as a result of intracellular calcium store depletion in mouse anococcygeus cells. Since the non-selective cation current appears to underlie the calcium entry process responsible for maintaining the sustained contractions to agonists in this tissue, this action of SNP may represent an important mechanism by which nitrates relax non-vascular smooth muscle.

Two distinct membrane currents activated by cyclopiazonic acid‐induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus

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. Voltage-dependent 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 microM) 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 microM) induced no further inward current. 5. In organ bath studies, CPA (10 microM)-induced contractions of the mouse anococcygeus were inhibited by cadmium and nickel (both 50-400 microM) and the general calcium entry blocker, SKF 96365 (10 microM); lanthanum and gadolinium had no effect at concentrations up to 400 microM. The pharmacology of the CPA-induced non-selective cation current mirrored that of the CPA-induced whole muscle contraction being reversed by cadmium (100 microM) and SKF 96365 (10 microM), but unaffected by lanthanum (400 microM). 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.

Inhibition of relaxations to nitrergic stimulation of the mouse anococcygeus by duroquinone.

1. The role of copper/zinc superoxide dismutase (Cu/Zn SOD) in protection of nitrergic neurotransmission in the mouse anococcygeus was investigated by use of duroquinone (DQ), which generates superoxide anions within tissues via reduction by flavoprotein enzymes. 2. In control anococcygeus muscles, DQ (10-100 microM) produced concentration-related inhibition (-log IC40 = 4.41) of relaxations to exogenous nitric oxide (NO; 15 microM). Nitrergic relaxations induced by field stimulation (10 Hz; 10 s train) were much less affected, 100 microM DQ reducing nitrergic relaxations by only 14 +/- 6%. 3. Following incubation with the Cu/Zn SOD inhibitor, diethyldithiocarbamate (DETCA; 3 mM; 45 min incubation; 10 min washout), the inhibitory effects of DQ on relaxations to NO were potentiated (-log IC40 = 5.22), and clear, concentration-related inhibitions of nitrergic relaxations were now observed (-log IC40 = 4.54). In both cases, these inhibitions were partially reversed by Cu/Zn SOD (250 u ml-1). In DETCA-treated tissues, DQ (100 microM) also reduced relaxations to sodium nitroprusside (1 microM) and S-nitroso-glutathione (30 microM), but potentiated those to 8-Br-cyclic GMP (100 microM). 4. Neither hydroquinone (HQ: 100 microM) nor 1,4-benzoquinone (BQ: 100 microM), both of which reduced responses to exogenous NO, inhibited relaxations induced by field stimulation in DETCA-treated tissues. Indeed, when added during DQ-induced inhibition of nitrergic relaxations, both HQ and BQ produced partial reversal of the block. 5. DQ had no effect on the detection of superoxide anions estimated via the xanthine:xanthine oxidase chemiluminescence assay, or of authentic NO as measured by a chemical microsensor. However, the detection of both superoxide anions and NO in these assays was inhibited by inclusion of either HQ or BQ. 6. The results support the proposal that nitrergic transmission in the peripheral nervous system is protected by Cu/Zn SOD activity in the region of the neuroeffector junction, and this may explain the lack of effect of superoxide anion generating drugs such as DQ. Such an explanation does not hold for either HQ or BQ, which appear to be acting directly as free radical scavengers in these experiments.

Duroquinone, benzoquinone and nitrergic relaxation of the mouse anococcygeus muscle

Duroquinone, benzoquinone and nitrergic relaxation of the mouse anococcygeus muscle

Variable potency of nitrergic‐nitrovasodilator relaxations of the mouse anococcygeus against different forms of induced tone

1. U46619 (thromboxane A2 receptors; 0.002-1 microM), carbachol (muscarinic M3 receptors; 0.1-100 microM), cyclopiazonic acid (CPA; Ca(2+)-ATPase inhibitor; 0.1-30 microM) and K+ (5-100 mM) produced concentration-dependent contractions of the mouse isolated anococcygeus muscle. Equi-effective, submaximal concentrations of each agent were used in further experiments (40 nM U46619; 5 microM carbachol; 5 microM CPA; 70 mM K+). 2. Nifedipine (1 microM) totally abolished contractile responses to K+; those to U46619, carbachol and CPA were reduced by only 20-30% in the presence of nifedipine, but were greatly reduced (> 90%) by a combination of nifedipine and SKF 96365 (0.1-40 microM). 3. In Ca(2+)-free medium, contractions to K+ and CPA were abolished. Small residual responses remained to both carbachol and U46619; those to carbachol were transient, could not be repeated in the continued absence of Ca2+ and were prevented by pre-incubation with CPA, but unaffected by SKF 96365; those to U46619 were sustained, could be repeated in the absence of Ca2+, and were resistant to CPA and SKF 96365. 4. Tone induced by all four agents could be relaxed by sodium nitroprusside (SNP), but with a clear order of potency. SNP (pIC40) was most effective against U46619 (7.92), less so against carbachol (6.80) and CPA (6.68), and least potent against K+ (5.94). A similar order of potency was observed with 8Br-cyclic GMP (50 microM) and nitrergic field stimulation (1-20 Hz). 5. The relaxant potency of SNP was similar in normal Krebs solution and in high K+ (70 mM) Krebs containing 1 microM nifedipine. 6. Inclusion of SNP (0.01-1 microM) or 8Br-cyclic GMP (50 microM) in the Ca2+-free medium inhibited the transient residual response to carbachol. Inclusion of similar concentrations of SNP or 8Br-cyclic GMP,during Ca2+ re-loading, increased the subsequent residual contraction to carbachol in Ca2+-free medium.7. At higher concentrations, SNP (0.1-10 microM) produced a partial relaxation of the sustained contraction to U46619 in Ca2+-free medium.8. Thus, the relaxant potency of the nitrergic stimuli was dependent on the agent and mechanism used to induce tone in the preparation. Examination of the contractile/relaxant interactions suggests that altered Ca2+ sequestration and inhibition of contractile protein function may underlie nitrergic relaxations of this tissue.

L‐368,899, a potent orally active oxytocin antagonist for potential use in preterm labor

AbstractL‐368,899 [1S‐(‐7,7‐dimethyl‐2‐endo‐(2S‐amino‐4‐(methylsulfonyl)butyramido)‐bicyclo(2.2.1)‐heptan‐1‐yl)methanesulfonyl)‐4‐(2‐methylphenyl)piperazine] was characterized in vitro and in vivo as a potent and selective, orally bioavailable oxytocin (OT) antagonist. L‐368,899 exhibits high affinity for rat (Ki, 3.6 nM) and human (Ki, 13 nM) uterine OT receptors with selectivity versus liver arginine‐vasopressin (AVP)‐V1 and kidney AVP‐V2 receptors in both species. In vitro, L‐368,899 is a potent and competitive OT antagonist in the rat isolated uterus (pA2, 8.9) and mouse anococcygeus muscle (pA2, 8.3) and is inactive against a number of different contractile agents in these preparations. L‐368,899 also inhibits OT‐stimulated phosphatidylinositol turnover in rat uterine slices and exhibits no agonist‐like activity in any of the in vitro assays. In vivo, L‐368,899 selectively antagonizes the uterine contractile effects of OT in the anesthetized rat given both i.v. and intraduodenally (i.d.) (AD50; 350 μg/kg i.v., 7 mg/kg i.d.) with a moderate duration of action. In near‐term pregnant rhesus macaques, L‐368,899 is a potent inhibitor of OT‐stimulated uterine activity (AD50, 27 μg/kg i.v.) and OT‐mediated spontaneous, nocturnal uterine contractions. L‐368,899 is orally bioavailable in several species and, combined with adequate aqueous solubility, represents a potential new tocolytic agent for both oral and i.v. use. © 1993 wiley‐Liss, Inc.

Localisation of nitric oxide synthase within non-adrenergic, non-cholinergic nerves in the mouse anococcygeus

Immunocytochemical staining of whole mount preparations of the mouse anococcygeus muscle, using antibodies to rat brain nitric oxide synthase (NOS), revealed a dense network of NOS-immunoreactive nerve fibres running through the tissue. These fibres were resistant to the sympathetic neurotoxin 6-hydroxydopamine and are therefore likely to be the non-adrenergic nerves which mediate relaxation of this smooth muscle. Further, NOS-immunoreactive fibres were absent following denervation by cold-storage (4°;C; 72 h), which has been shown to abolish non-adrenergic, non-cholinergic (NANC) relaxations. The results provide strong support for the hypothesis that the l-arginine:NO pathway is responsible for the generation of the NANC transmitter in the anococcygeus.