Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03–30 μM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded.
2. Sodium rhein (0.3, 3 and 30 μM) significantly potentiated (52±11% at 30 μM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 μM) or ω-conotoxin GVIA (10 nM) sodium rhein (3 and 30 μM) did not enhance, but significantly reduced (49±10% and 44±8%, respectively, at 30 μM) acetylcholine-induced contractions.
3. Sodium rhein (0.3, 3 and 30 μM) significantly increased (65±11% at 30 μM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 μM), ω-conotoxin GVIA (10 nM) or atropine (0.1 μM), sodium rhein (3 and 30 μM) significantly reduced (50±10%, 55±8% and 46±10%, respectively, at 30 μM) substance P-induced contractions.
4. N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 μM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30±6%)- or substance P (36±6%)-induced contractions.
5. Sodium rhein (30 μM) significantly potentiated (29±9%) the electrically-evoked contractions. L-NAME (100 μM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 μM) significantly increased (32±9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment.
6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca²⁺ entry through the N-type Ca²⁺ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.

     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Involvement of vasoactive intestinal polypeptide in nicotine-induced relaxation of the rat gastric fundus

1. Nicotine-induced relaxation and release of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-like immunoreactivity (LI) were measured in longitudinal muscle strips from the rat gastric fundus.
2. Under non-cholinergic conditions (0.3 μM atropine), nicotine (3–300 μM) produced concentration-dependent relaxations of the 5-hydroxytryptamine (3 μM)-precontracted strips. Under non-adrenergic non-cholinergic (NANC) conditions (0.3 μM atropine+1 μM phentolamine+1 μM nadolol), relaxations induced by sub-maximal nicotine concentrations (10 and 30 μM) were significantly smaller, while that produced by the highest concentration used (300 μM) was similar to that seen under non-cholinergic conditions.
3. Re-exposure to the same nicotine concentration 1 h later induced smaller relaxations, indicating desensitization. The reductions seen in the second responses were proportional to the concentration used.
4. Under non-cholinergic conditions, the relaxant response to 30 μM nicotine was abolished by hexamethonium (100 μM) and significantly reduced by tetrodotoxin (TTX, 3 μM). The TTX-resistant component was not observed under NANC conditions.
5. NANC relaxation induced by 30 μM nicotine was significantly reduced by a specific anti-VIP serum (approximately 35% less than that seen with normal rabbit serum).
6. Nicotine (30–300 μM) caused significant, concentration-dependent increases in the outflow of VIP- and PHI-LI from the strips; these effects were also diminished with re-exposure. The increases in both types of immunoreactivity evoked by nicotine (300 μM) were abolished by hexamethonium (300 μM), TTX (3 μM) and a calcium-free medium.
7. These findings indicate that VIP and possibly PHI are involved in NANC relaxation of the rat gastric fundus induced by nicotine.

     

Effects of L-NG-nitro-arginine on noradrenaline induced contraction in the rat anococcygeus muscle

1. The influence of L-N^G-nitro-arginine (L-NOARG, 30 μM) on contractile responses to exogenous noradrenaline was studied in the rat anococcygeus muscle.
2. Noradrenaline (0.1–100 μM) contracted the muscle in a concentration-dependent manner. L-NOARG (30 μM) had no effect on noradrenaline responses.
3. Phenoxybenzamine (Pbz 0.1 μM) depressed by 46% (P<0.001) the maximum response and shifted to the right (P<0.001) the E/[A] curve to noradrenaline (pEC₅₀ control: 6.92±0.09; pEC₅₀ Pbz: 5.30±0.10; n=20).
4. The nested hyperbolic null method of analysing noradrenaline responses after phenoxybenzamine showed that only 0.61% of the receptors need to be occupied to elicit 50% of the maximum response, indicating a very high functional receptor reserve.
5. Contractile responses to noradrenaline after partial α₁-adrenoceptor alkylation with phenoxybenzamine (0.1 μM) were clearly enhanced by L-NOARG.
6. The potentiating effect of L-NOARG on noradrenaline responses after phenoxybenzamine was reversed by (100 μM) L-arginine but not by (100 μM) D-arginine.
7. These results indicate that spontaneous release of NO by nitrergic nerves can influence the α₁-adrenoceptor-mediated response to exogenous noradrenaline.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Recovery by ascorbate of impaired nitric oxide-dependent relaxation resulting from oxidant stress in rat aorta

1. In this study we investigated the ability of ascorbate to protect nitric oxide from destruction by superoxide anion.
2. Ascorbate produced concentration-dependent relaxation of rings of rat aorta, comprising two components: the first, seen at 1–300 μM, reached a maximum of 45.3±2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 μM), demonstrating involvement of nitric oxide. The second occurred at concentrations of 1 mM and above and was associated with falls in the pH of the bathing fluid.
3. Pretreatment with ascorbate at concentrations up to 3 mM had no effect on the relaxation to acetylcholine (10 nM–10 μM) on endothelium-containing rings or adenosine (0.1 μM–3 mM) on endothelium-denuded rings.
4. An oxidant stress was applied to aortic rings, comprising inhibition of endogenous Cu/Zn superoxide dismutase by diethyldithiocarbamate (0.1 mM) followed by generation of superoxide anion by hypoxanthine (0.1 mM/xanthine oxidase (16 u ml⁻¹). This reduced maximal acetylcholine-induced relaxation from 96.7±1.3% to 42.4±3.5% (P<0.001). Treatment with ascorbate (30 μM–3 mM) reversed this blockade in a concentration-dependent manner.
5. Our findings show that ascorbate has the ability to protect nitric oxide from destruction by superoxide anion. This action is seen with ascorbate at levels normally present in plasma, suggesting that this antioxidant may exert a tonic protective effect on nitric oxide within the vasculature.

     

Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat

1. Intracellular recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus (LC). The pressure application of α,β-methylene ATP (α,β-meATP) caused reproducible depolarizations which were depressed by suramin (30 μM) and abolished by suramin (100 μM). Pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 10, 30 μM) also concentration-dependently inhibited the α,β-meATP-induced depolarization, although with a much slower time-course than suramin. Almost complete inhibition developed with 30 μM PPADS. Reactive blue 2 (30 μM) did not alter the effect of α,β-meATP, while reactive blue 2 (100 μM) slightly depressed it.
2. Pressure-applied (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also depolarized LC neurones. Kynurenic acid (500 μM) depressed and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) abolished the response to AMPA. Suramin (100 μM) potentiated the AMPA effect.
3. Pressure-applied noradrenaline hyperpolarized LC neurones. Suramin (100 μM) did not alter the effect of noradrenaline.
4. Focal electrical stimulation evoked biphasic synaptic potentials consisting of a fast depolarization (p.s.p.) followed by a slow hyperpolarization (i.p.s.p.). A mixture of D(−)-2-amino-5-phosphonopentanoic acid (AP-5; 50 μM), CNQX (50 μM) and picrotoxin (100 μM) depressed both the p.s.p. and the i.p.s.p. Under these conditions suramin (100 μM) markedly inhibited the p.s.p., but did not alter the i.p.s.p. In the combined presence of AP-5 (50 μM), CNQX (50 μM), picrotoxin (100 μM), strychnine (0.1 μM), tropisetron (0.5 μM) and hexamethonium (100 μM), a high concentration of suramin (300 μM) almost abolished the p.s.p. without changing the i.p.s.p.
5. In the presence of kynurenic acid (500 μM) and picrotoxin (100 μM), PPADS (30 μM) depressed the p.s.p. Moreover, the application of suramin (100 μM) to the PPADS (30 μM)-containing medium failed to cause any further inhibition. Neither PPADS (30 μM) nor suramin (100 μM) altered the i.p.s.p.
6. It was concluded that the cell somata of LC neurones are endowed with excitatory P2-purinoceptors. ATP may be released either as the sole transmitter from purinergic neurones terminating at the LC or as a co-transmitter of noradrenaline from recurrent axon collaterals or dendrites of the LC neurones themselves.

     

Evidence that both nitric oxide (NO) and a non-NO hyperpolarizing factor elicit NANC nerve-mediated relaxation in the rat isolated anococcygeus

1. Responses to electrical field stimulation (EFS; 0.5–10 Hz, 0.2 ms duration, supramaximal voltage for 20 s) of non-adrenergic, non-cholinergic, (NANC) nerves were obtained in preparations of rat anococcygeus pre-contracted with titrated concentrations of phenylephrine (0.1–1 μM) to ∼40% of their maximum contraction to phenylephrine (F_max) regardless of drug treatment.
2. With this set level of active force, NANC nerve stimulation resulted in relaxations that were maximal (peak relaxation) at 0.5–1 Hz, abolished by tetrodotoxin (1 μM) but only minimally blocked by the nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine, (L-NOARG; 100 μM). Furthermore, the nitric oxide (NO) scavenger, oxyhaemoglobin (HbO; 30 μM) gave no further block alone or in combination with L-NOARG (100 μM). By comparison, in preparations contracted with phenylephrine to ∼70% F_max, relaxations to NANC nerve stimulation were markedly reduced or abolished by combined treatment with L-NOARG (100 μM) and HbO (30 μM).
3. Nifedipine (0.3 μM) significantly inhibited NANC nerve-mediated relaxations, which became frequency-dependent and abolished those resistant to L-NOARG (100 μM) and HbO (30 μM).
4. These data suggest that a non-NO, hyperpolarizing factor and NO both contribute to NANC nerve-mediated inhibitory responses in the rat anococcygeus. However, responses to the non-NO factor were observed only in preparations contracted sub-maximally by a nifedipine-sensitive mechanism.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[³H]-arginine to L-[³H]-citrulline.
2. Elevation of extracellular K⁺ to 20, 35 or 60 mM concentration-dependently augmented L-[³H]-citrulline production. The response to 35 mM KCl was abolished by N^G-nitro-L-arginine (100 μM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl₂ concentration up to 10 mM also prevented the K⁺ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity.
3. However, the effect of high K⁺ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine.
4. The 35 mM K⁺ response was insensitive to ω-conotoxin GVIA (1 μM) and nifedipine (100 μM), but could be prevented in part by ω-agatoxin IVA (0.1 and 1 μM). The inhibition caused by 0.1 μM ω-agatoxin IVA (∼30%) was enhanced by adding ω-conotoxin GVIA (1 μM) or nifedipine (100 μM). Further inhibition (up to above 70%) could be observed when the three Ca²⁺ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 μM) the K⁺-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 μM ω-conotoxin GVIA plus 100 μM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX.
5. It was concluded that elevation of extracellular [K⁺] causes NO synthase activation by external Ca⁺ entering cells mainly through channels of the P/Q-type. Other Ca²⁺ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.

     

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline- and 5-hydroxytryptamine (5-HT)-induced pressor responses of the rat perfused isolated mesenteric vascular bed.
2. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose-dependent manner. Nifedipine (1 μM) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31±5%. Niflumic acid (10 and 30 μM) also inhibited the noradrenaline-induced increase in perfusion pressure and 30 μM niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34±6%.
3. The increases in perfusion elicited by 5-HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 μM) in a concentration-dependent manner and 30 μM niflumic acid inhibited responses to 0.3 and 3 nmol 5-HT by, respectively, 49±8% and 50±7%. Nifedipine (1 μM) decreased the pressor response to 3 nmol 5-HT by 44±9%.
4. In the presence of a combination of 30 μM niflumic acid and 1 μM nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5-HT (3 nmol) was not significantly greater than with niflumic acid (30 μM) alone. Thus the effects of niflumic acid and nifedipine were not additive.
5. In Ca-free conditions the transient contractions induced by 5-HT (3 nmol) were not reduced by 30 μM niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5-HT to its receptor.
6. Niflumic acid 30 μM did not inhibit the pressor responses induced by KCl (20 and 60 μmol) which were markedly reduced by 1 μM nifedipine. In addition, 1 μM levcromakalim decreased pressor responses produced by 20 μmol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels.
7. It is concluded that niflumic acid selectively reduces a component of noradrenaline- and 5-HT-induced pressor responses by inhibiting a mechanism which leads to the opening of voltage-gated calcium channels. Our data suggest that the Ca²⁺-activated chloride conductance may play a pivotal role in the activation of voltage-gated calcium channels in agonist-induced constriction of resistance blood vessels.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones

1. The interactions between N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) were investigated in striatal slices, by utilizing intracellular recordings, both in current- and voltage-clamp mode.
2. Bath-application (50 μM) or focal application of NMDA induced a transient membrane depolarization, while in the voltage-clamp mode, NMDA (50 μM) caused a transient inward current. Following bath-application of the non-selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), NMDA responses were reversibly potentiated both in current (197±15% of control) and voltage-clamp experiments (200±18% of control).
3. Bath-application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG, 10–300 μM) resulted in a dose-dependent potentiation of NMDA-induced membrane depolarization (up to 400±33% of control). This potentiation was either prevented by preincubation with (RS)-α-methyl-4-carboxyphenylglycine (RS-α-MCPG, 300 μM), or blocked when applied immediately after 3,5-DHPG wash-out.
4. Neither (2S,1′S,2′S)2-(2′-carboxycyclopropyl)glycine (L-CCG I, up to 100 μM) nor (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV, 1 μM), agonists for group II mGluRs caused any change in NMDA responses. Likewise, L-serine-O-phosphate (L-SOP, 30 μM), agonist for group III mGluRs, did not affect the NMDA-induced depolarization.
5. The enhancement of the NMDA responses was mimicked by phorbol-12,13-diacetate (PDAc, 1 μM) which activates protein kinase C (PKC). The 3,5-DHPG-mediated potentiation of the NMDA-induced depolarization was prevented by preincubation with staurosporine (100 nM) or calphostin C (1 μM), antagonists of PKC.
6. Electrophysiological responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor activation were not affected by agonists for the three-classes of mGluRs.
7. The present data suggest that group I mGluRs exert a positive modulatory action on NMDA responses, probably through activation of PKC. This functional interaction in the striatum appears of crucial importance in the understanding of physiological and pathological events, such as synaptic plasticity and neuronal death, respectively.

     

Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle.
2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO₂Me) and a recently developed human chymase substrate Pro¹¹-,D-Ala¹²)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used.
3. Angiotensin I (AI) (0.3 μM) induced a contractile response amounting to 58±5% (n=12) of the initial K⁺ (124 mM)-induced contractions. This response was reduced to 36±3% (n=8) by the ACE-inhibitor enalaprilat (10 μM), while pretreatment with soybean trypsin inhibitor (STI 200 μg ml⁻¹) or CH 5450 (10 μM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19±5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1±1% (n=6).
4. The substrate (Pro¹¹-,D-Ala¹²)-AI (3 μM) produced contractions which amounted to 57±4% (n=13) of the initial K⁺ (124 mM) contractions. These contractions were not affected by enalaprilat (10 μM). On the other hand, STI (200 μg ml⁻¹) and CH 5450 (10 μM) added separately, depressed the (Pro¹¹-,D-Ala¹²)-AI-induced contractions to 34±5% (n=6) and 24±4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition.
5. Experiments with detrusor membrane fractions incubated with AI (50 μM) were performed. In the presence of enalaprilat (100 μM), carboxypeptidase inhibitor CPI (10 μg ml⁻¹) and aprotinin (15 μM), CH 5450 (10 nM–1 μM) caused a concentration-dependent inhibition of AII formation.
6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells

1. The effect of increasing cellular tyrosine phosphorylation by inhibiting endogenous tyrosine phosphatases was examined on voltage-operated calcium channel currents in vascular smooth muscle cells.
2. In single ear artery smooth muscle cells of the rabbit, studied by the whole cell voltage clamp technique, intracellular application of the tyrosine phosphatase inhibitors, sodium orthovanadate (100 μM) and peroxyvanadate (100 μM orthovanadate+1 mM H₂O₂) increased voltage-operated calcium channel currents by 56% and 83%, respectively.
3. Bath application of two other membrane permeant tyrosine phosphatase inhibitors, phenylarsine oxide (100 μM) and dephostatin (50 μM) also increased voltage-operated calcium channel currents by 48% and 52%, respectively.
4. The selective tyrosine kinase inhibitor, tyrphostin-23 (100 μM) reduced calcium channel currents by 41%. Pre-incubation with tyrphostin-23 abolished the effects of peroxyvanadate, phenylarsine oxide and dephostatin on calcium channels.
5. Western blot analysis of rabbit ear artery cell lysates showed increased tyrosine phosphorylation of several endogenous proteins following treatment with peroxyvanadate.
6. These results indicate that a number of structurally dissimilar inhibitors of tyrosine phosphatases increase voltage-operated calcium channel currents in arterial smooth muscle cells presumably due to increased tyrosine phosphorylation.

     

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 μM–3 mM) produced concentration-dependent, well-sustained contraction. Its −logEC₅₀ was 3.74±0.05 (mean±s.e.mean) and its maximal effect was equivalent to 97.5±4.2% of the response to acetylcholine (ACh, 1 mM).
2. Vanadate (200 μM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 μM), zileuton (10 μM), a mixture of atropine, mepyramine and phentolamine (each at 1 μM), or by mast cell degranulation with compound 48/80.
3. Vanadate (200 μM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 μM) or to a Ca²⁺-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 μM) in Ca²⁺-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca²⁺-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca²⁺ stores. In such tissues cyclopiazonic acid (CPA; 10 μM) prevented Ca²⁺-induced recovery of vanadate-induced contraction.
4. Tissue incubation in K⁺-rich (80 mM) PSS, K⁺-free PSS, or PSS containing ouabain (10 μM) did not alter vanadate (200 μM)-induced contraction. Ouabain (10 μM) abolished the K⁺-induced relaxation of human bronchus bathed in K⁺-free PSS. This action was not shared by vanadate (200 μM). The tissue content of Na⁺ was increased and the tissue content of K⁺ was decreased by ouabain (10 μM). In contrast, vanadate (200 μM) did not alter the tissue content of these ions. Tissue incubation in a Na⁺-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 μM).
5. Vanadate (200 μM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 μM), staurosporine (1 μM) and calphostin C (1 μM). Genistein (100 μM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate.
6. Vanadate (0.1–3 mM) and ACh (1 μM–3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca²⁺-free medium either alone or in combination with ryanodine (10 μM).
7. In human cultured tracheal smooth muscle cells, histamine (100 μM) and vanadate (200 μM) each produced a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i).
8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 μM) in human bronchus was associated with cellular depolarization.
9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca²⁺ from an intracellular store. The Ca²⁺ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca²⁺]_i that are close to basal.

     

The signalling pathway which causes contraction via P2-purinoceptors in rat urinary bladder smooth muscle

1. The signalling pathway which causes contractions to adenosine 5′-O-2-thiodiphosphate (ADPβS) and α,β-methylene adenosine 5′-diphosphate (α,β-Me ADP) was investigated in rat urinary bladder smooth muscle by measuring isotonic tension.
2. The responses to 10 μM α,β-methylene adenosine 5′-triphosphate (α,β-Me ATP) in 0 and 3.6 mM Ca²⁺ were 5.9±1.3 (n=10) and 122.2±6.4 (n=8) % respectively of those obtained in 1.8 mM Ca²⁺, whereas those to 100 μM ADPβS were 34.6±3.3 (n=8) and 96.8±7.2 (n=8) %, in 0 and 3.6 mM Ca²⁺, respectively. In both experimental conditions, the responses to the two agonists expressed as % of the control responses were significantly different (P<0.01).
3. Indomethacin at high concentrations (>1 μM) decreased the responses to α,β-Me ATP (10 μM), ADPβS (100 μM) and α,β-Me ADP (100 μM). However, no significant difference was obtained between the responses to all the agonists at 30 μM indomethacin.
4. 2-Nitro-4-carboxphenyl n,n-diphenylcarbamate (NCDC) at concentrations between 1 μM and 100 μM concentration-dependently decreased the responses to ADPβS (100 μM) and α,β-Me ADP (100 μM) and almost completely inhibited them at 100 μM. Although the responses to α,β-Me ATP (10 μM) were also inhibited by the drug, at 50 and 100 μM NCDC the responses to α,β-Me ATP were significantly larger than those to ADPβS and α,β-Me ADP (P<0.01).
5. NCDC 100 μM significantly inhibited the KCl-induced contraction to 65.9±4.9% (n=6) of the control (P<0.01).
6. It is suggested that the contraction via ADPβS-sensitive receptors in the rat urinary bladder smooth muscle mainly depends on Ca²⁺ ions liberated from intracellular Ca²⁺ stores, though the contribution of Ca²⁺ ions from the extracellular space cannot be neglected. The release of Ca²⁺ ions from stores is mainly mediated by the production of inositol trisphosphate (IP₃) via the activation of phospholipase C.