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.

     

Relaxation of corpus cavernosum and raised intracavernous pressure by berberine in rabbit

1. In the present study, we have investigated the effect of berberine in rabbit isolated corpus cavernosum and measured the intracavernous pressure (ICP) change after intracavernosal injection of berberine in rabbit.
2. Berberine alone suppressed the basal tone and induced a concentration (0.1–100 μM)-dependent relaxation in phenylephrine (PE)-precontracted corpus cavernosum.
3. Tetrodotoxin (0.1 and 1 μM) treatment had no significant effect on the berberine-induced relaxation. Phentolamine (1 and 10 μM), propranolol (1 and 3 μM) and atropine (1 and 3 μM) were also without effect. These results suggest that berberine might cause relaxation of the cavernosal strip by direct action on the corpus cavernosum, not by a neuronal effect. Furthermore, muscarinic- and β-adrenoceptors were not involved.
4. Berberine-induced relaxations were significantly reduced by endothelium removal and by exposure to L-N^G-nitro arginine methyl ester (0.1 and 0.3 mM), but not indomethacin (30 μM).
5. In endothelium-deprived corpus cavernosal tissues, berberine-induced relaxations were significantly reduced in high K⁺ medium (KCl=60 mM), by charybdotoxin (ChTX) and 4-aminopyridine (4-AP) but not by glibenclamide and apamin.
6. After intracavernous injection of berberine (1, 2, 3 and 5 mg kg⁻¹), the ICP rose from 12.7±3.6 to 13.2±5.4, 25.3±6.1, 46.5±8.2, and 63.4±10.2 mmHg, respectively. The duration of tumescence ranged from 11.5–43.7 min.
7. The results show that berberine possesses a relaxant effect on rabbit corpus cavernosal tissues which is attributable to both endothelium-dependent and-independent properties. While the former component is apparently due to the release of NO from sinusoidal endothelium, the endothelium-independent mechanism involved in berberine relaxation is probably linked to ChTX- and 4-AP-sensitive K⁺ channel activation in the cavernosal vasculature.

     

The actions of the cannabinoid receptor antagonist,SR 141716A,in the rat isolated mesenteric artery

1. The actions of the cannabinoid receptor antagonist, SR 141716A, were examined in rat isolated mesenteric arteries. At concentrations greater than 3 μM, it caused concentration-dependent, but endothelium-independent, relaxations of both methoxamine- and 60 mM KCl-precontracted vessels.
2. SR 141716A (at 10 μM, but not at 1 μM) inhibited contractions to Ca²⁺ in methoxamine-stimulated mesenteric arteries previously depleted of intracellular Ca²⁺ stores. Neither concentration affected the phasic contractions induced by methoxamine in the absence of extracellular Ca²⁺.
3. SR 141716A (10 μM) caused a 130 fold rightward shift in the concentration-response curve to levcromakalim, a K⁺ channel activator, but had no effect at 1 μM.
4. SR 141716A (10 μM) attenuated relaxations to NS 1619 (which activates large conductance, Ca²⁺-activated K⁺ channels; BK_Ca). The inhibitory effect of SR 141716A on NS 1619 was not significantly different from, and was not additive with, that caused by a selective BK_Ca inhibitor, iberiotoxin (100 nM). SR 141716A (1 μM) did not effect NS 1619 relaxation.
5. SR 141716A (10 μM) had no effect on relaxations to the nitric oxide donor S-nitroso-N-acetylpenicillamine, or relaxations to carbachol in the presence of 25 mM KCl.
6. The results show that, at concentrations of 10 μM and above, SR 141716A causes endothelium-independent vasorelaxation by inhibition of Ca²⁺ entry. It also inhibits relaxations mediated by K⁺ channel activation. This suggests that such concentrations of SR 141716A are not appropriate for investigation of cannabinoid receptor-dependent processes.

     

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 μM) produced concentration-related contractions of the mouse isolated anococcygeus muscle; 0.2 μM 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-N^G-nitro-arginine (L-NOARG; 100 μM), the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 μM), or a combination of these two drugs, relaxations to field stimulation were abolished at all frequencies studied. Relaxations to sodium nitroprusside (0.01–5 μM) were unaffected by L-NOARG but strongly inhibited by ODQ; neither enzyme inhibitor affected relaxations to 8-Br-cyclic GMP (10 μM).
4. Nifedipine (1 μM) reduced the contractile response to 0.2 μM 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.

     

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.

     

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.

     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

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.

     

Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices

1. Responses in brain produced by the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [³H]-noradrenaline from prisms of rat hippocampus.
2. AMPA (1–1000 μM) stimulated the release of [³H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 μM), LY 293558 (1 and 10 μM) and GYKI 52466 (10 and 30 μM).
3. By use of an experimental procedure with consecutive applications of AMPA (100 μM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1–300 μM). The concentration-response curve for AMPA-induced release of [³H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus.
4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 μM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was long-lasting (second application of cyclothiazide was ineffective).
5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [³H]-noradrenaline: forskolin (10 μM, +78%), Rp-cAMPS (100 μM, +65%), Ro 31-8220 (10 μM, + 163%) and thapsigargin (100 μM, +161%).
6. The AMPA receptor-mediated response regulating the release of [³H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca²⁺ stores.

     

S-nitrosothiols and the nitrergic neurotransmitter in the rat gastric fundus: effect of antioxidants and metal chelation

1. The effects of the antioxidants ascorbic acid and α-tocopherol and of the metal chelator ethylenediaminetetraacetic acid (EDTA) were studied on relaxations in response to S-nitrosothiols, authentic nitric oxide (NO) and nitrergic non-adrenergic non-cholinergic stimulation of the rat gastric fundus.
2. The S-nitrosothiols S-nitrosocysteine (1–100 nM), S-nitrosoglutathione (0.01–3 μM) and S-nitroso-N-acetylpenicillamine (0.01–3 μM) induced concentration-dependent relaxations of the rat gastric fundus muscle strips, which were precontracted with prostaglandin F_2α. The relaxations to all S-nitrosothiols were concentration-dependently enhanced by the antioxidants ascorbic acid (0.1–3 μM) and α-tocopherol (3–30 μM) and inhibited by the metal chelator EDTA (26 μM).
3. Ascorbic acid and α-tocopherol alone did not induce a relaxation of the precontracted rat gastric fundus muscle strip. However, when ascorbic acid (1 μM) or α-tocopherol (1 μM) were injected in the organ bath 1 minute after S-nitrosoglutathione (0.1 μM) or after S-nitroso-N-acetylpenicillamine (0.1 μM), they induced an immediate, sharp and transient relaxation. This relaxation was inhibited by the superoxide generator pyrogallol (2 μM). Such a relaxation to ascorbic acid or α-tocopherol was not observed in the presence of S-nitrosocysteine (10 nM).
4. Electrical field stimulation (0.5–4 Hz) of the precontracted rat gastric fundus strips induced frequency-dependent nitrergic relaxations which were mimicked by authentic NO (3–300 nM) and by acidified sodium nitrite NaNO₂ (0.3–10 μM). Ascorbic acid (0.3–3 μM), α-tocopherol (3–30 μM) or EDTA (26 μM) did not affect the relaxations to nitrergic stimulation, NO or NaNO₂.
5. In summary, relaxations to S-nitrosothiols in the rat gastric fundus are enhanced by the antioxidants ascorbic acid and α-tocopherol and inhibited by the metal chelator EDTA. However, relaxations to nitrergic stimulation of the rat gastric fundus or those to authentic NO were not affected by the antioxidants or by the metal chelator. These results indicate that antioxidants and metal chelators have a different effect on the biological activity of S-nitrosothiols and on that of the nitrergic neurotransmitter. Therefore, our results suggest that S-nitrosothiols do not act as intermediate compounds in nitrergic neurotransmission in the rat gastric fundus.

     

ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia

1. ATP (10–100 μM), but not glutamate (100  μM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 μM) nor glutamate (100 μM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 μM), which is metabolized in the cytosol to BAPTA (an intracellular Ca²⁺ chelator), completely inhibited the plasminogen release evoked by ATP (100 μM). The Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 induced plasminogen release in a concentration-dependent manner (0.3 μM to 10 μM).
2. ATP induced a transient increase in the intracellular calcium concentration ([Ca²⁺]_i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 μM) had no effect on [Ca²⁺]_i (70 out of 70 cells) in microglial cells. A second application of ATP (100 μM) stimulated an increase in [Ca²⁺]_i similar to that of the first application (21 out of 21 cells).
3. The ATP-evoked increase in [Ca²⁺]_i was totally dependent on extracellular Ca²⁺, 2-Methylthio ATP was active (7 out of 7 cells), but α,β-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca²⁺]_i. Suramin (100 μM) was shown not to inhibit the ATP-evoked increase in [Ca²⁺]_i (20 out of 20 cells). 2′- and 3′-O-(4-Benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), a selective agonist of P2X₇ receptors, evoked a long-lasting increase in [Ca²⁺]_i even at 1 μM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5′-triphosphate-2′, 3′-dialdehyde (oxidized ATP, 100 μM), a selective antagonist of P2X₇ receptors, blocked the increase in [Ca²⁺]_i induced by ATP (10 and 100 μM).
4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca²⁺]_i via the ionotropic P2X₇ receptor which stimulates the release of plasminogen from the microglia.

     

Stretch-evoked inhibition of spontaneous migrating contractions in a whole mount preparation of the guinea-pig upper urinary tract

1. The effects of circumferentially-applied stretch on the spontaneous contractility of a whole mount preparation of the guinea-pig upper urinary tract (UUT) (renal pelvis and ureter) were investigated by use of standard isometric tension recording techniques.
2. Simultaneous tension recordings of the proximal and distal portions of the renal pelvis (RP) and ureter revealed that spontaneous contractions, in 79% (n=66) of preparations, originated in the proximal RP (at a frequency of 4.5 min⁻¹) and propagated to the distal RP and ureter at a velocity of 1–3 cm s⁻¹. Pretreatment with tetrodotoxin (TTX) (3–10 μM) or N^G-nitro-L-arginine (100 μM) had little effect on the spontaneous contractility of the UUT, motility indexes (MIs) (contraction amplitude×contraction frequency) calculated after 20 min exposure were little affected by TTX or N^G-nitro-L-arginine (L-NOARG). ω-Conotoxin GVIA (100 nM) significantly reduced MI values in both the proximal RP and ureter.
3. Exposure of the spontaneously-active UUT to capsaicin (10 μM for 15 min) induced a transient increase in UUT contractility, followed by a prolonged negative inotropic effect. The MI values, calculated 60 min after the washout of capsaicin, for the proximal and distal RP and ureter were reduced to 56%, 53% (n=18) and 61% (n=16), respectively, of their control values. This capsaicin pretreatment blocked the positive inotropic effects of transmural electrical nerve stimulation on UUT contractility to reveal a small inhibitory effect which was readily blocked by tetrodotoxin (3 μM) (n=3). The excitatory and inhibitory actions of nerve stimulation were both blocked by TTX (3 μM)
4. A second exposure to capsaicin (10 μM for 15 min), further reduced the MI values (calculated 60 min after washout) in the proximal and distal RP to 41% and 31%, respectively (n=6; P<0.05), of the initial control values.
5. In 61% (n=99) of preparations, the application of stretch to the proximal RP (0.5 to 2 mm) evoked a decrease in the amplitude of the contractions recorded in the distal RP, but not in the ureter. Stretch applied to the distal RP or ureter had no effect on the contractions recorded in the other regions of the UUT.
6. In 5 out of 6 preparations, a single application of capsaicin (10 μM for 15 min) had little effect on the change in contractile force of the distal RP evoked upon stretch of the proximal RP.
7. The inhibition of the distal RP upon stretch of the proximal RP was partially reduced (P<0.05) when the UUT was pretreated with the calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP (8–37) (1 μM).
8. The application of the CGRP receptor agonist, hCGRP (100 nM) inhibited contractility in the UUT in a region dependent manner. The MI of the proximal RP was decreased 32% after 6 min; while the MIs of the distal RP and ureter were reduced 83% and 63%, respectively, within 5 min of the application of hCGRP.
9. Glibenclamide (1 μM) had little effect on the spontaneous contractility of the UUT, but significantly reduced the inhibition of the distal RP evoked upon stretch (0.5 to 2 mm) of the proximal RP. TTX (3–10 μM), L-NOARG (100 μM) or ω-conotoxin GVIA (100 nM) had little effect on the stretch-evoked inhibition of the distal RP.
10. It was concluded that circumferential stretch of the proximal RP inhibits the contractility of the distal RP and that a component of this inhibition involves the activation of a glibenclamide-sensitive mechanism via the release of endogenous CGRP, possibly from the varicosities of intramural sensory nerves.

     

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine,barium, ouabain and 6-hydroxydopamine in chromaffin cells

1. Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μM) during 24 h, caused a concentration-dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154, did not enhance LDH release. Both lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (0.3–10 μM) decreased the veratridine-induced LDH release.
2. Penfluridol did not increase LDH release at concentrations 0.003–1 μM; 3–10 μM increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μM) did not protect against the cytotoxic effects of veratridine; at 1 μM, 15% protection was produced. Higher concentrations (3–10 μM) enhanced the cytotoxic effects of veratridine.
3. Ba²⁺ ions caused a concentration-dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μM lubeluzole and fully counteracted by 1 μM penfluridol. {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba²⁺.
4. Ouabain (10 μM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μM) and the lower concentrations of penfluridol (0.003–0.3 μM) prevented the ouabain cytotoxic effects. At higher concentrations (3 μM), penfluridol increased drastically the ouabain cytotoxic effects.
5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 μM. Lubeluzole (3–10 μM) or penfluridol (0.03–0.3 μM) had no cytoprotective effects against 6-OHDA.
6. Lubeluzole (3 μM), {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 (3 μM) and penfluridol (1 μM) blocked the current through Na⁺ channels in voltage-clamped chromaffin cells (I_Na) by around 20–30%. Ca²⁺ current through Ca²⁺ channels (I_Ca) was inhibited 57% by lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 were readily reversible.
7. Lubeluzole (3 μM) induced reversible blockade of the oscillations of the cytosolic Ca²⁺, [Ca²⁺]_i, in fura-2-loaded cells exposed to 30 or 100 μM veratridine. Penfluridol (1 μM) inhibited those oscillations in an irreversible manner.
8. The results suggest that lubeluzole and its R-isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na⁺ and Ca²⁺ entry, as well as the [Ca²⁺]_i oscillations. The Ba²⁺ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na⁺/Ca²⁺ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.

     

Possible mechanisms underlying the midazolam-induced relaxation of the noradrenaline-contraction in rabbit mesenteric resistance artery

1. The mechanisms underlying the midazolam-induced relaxation of the noradrenaline (NA)-contraction were studied by measuring membrane potential, isometric force and intracellular concentration of Ca²⁺([Ca²⁺]_i) in endothelium-denuded muscle strips from the rabbit mesenteric resistance artery. The actions of midazolam were compared with those of nicardipine, an L-type Ca²⁺-channel blocker.
2. Midazolam (30 and 100 μM) did not modify either the resting membrane potential or the membrane depolarization induced by 10 μM NA.
3. NA (10 μM) produced a phasic, followed by a tonic increase in both [Ca²⁺]_i and force. Midazolam (10–100 μM) did not modify the resting [Ca²⁺]_i, but attenuated the NA-induced phasic and tonic increases in [Ca²⁺]_i and force, in a concentration-dependent manner. In contrast, nicardipine (0.3 μM) attenuated the NA-induced tonic, but not phasic, increases in [Ca²⁺]_i and force.
4. In Ca²⁺-free solution containing 2 mM EGTA, NA (10 μM) transiently increased [Ca²⁺]_i and force. Midazolam (10–100 μM), but not nicardipine (0.3 μM), attenuated this NA-induced increase in [Ca²⁺]_i and force, in a concentration-dependent manner. However, midazolam (10 and 30 μM), had no effect on the increases in [Ca²⁺]_i and force induced by 10 mM caffeine.
5. In ryanodine-treated strips, which have functionally lost the NA-sensitive Ca²⁺- storage sites, NA slowly increased [Ca²⁺]_i and force. Nicardipine (0.3 μM) did not modify the resting [Ca²⁺]_i but partly attenuated the NA-induced increases in [Ca²⁺]_i and force. In the presence of nicardipine, midazolam (100 μM) lowered the resting [Ca²⁺]_i and further attenuated the remaining NA-induced increases in [Ca²⁺]_i and force.
6. The [Ca²⁺]_i-force relationship was obtained in ryanodine-treated strips by the application of ascending concentrations of Ca²⁺ (0.16–2.6 mM) in Ca²⁺-free solution containing 100 mM K⁺. NA (10 μM) shifted the [Ca²⁺]_i-force relationship to the left and enhanced the maximum Ca²⁺-induced force. Under these conditions, whether in the presence or absence of 10 μM NA, midazolam (10 and 30 μM) attenuated the increases in [Ca²⁺]_i and force induced by Ca²⁺ without changing the [Ca²⁺]_i-force relationship.
7. It was concluded that, in smooth muscle of the rabbit mesenteric resistance artery, midazolam inhibits the NA-induced contraction through its inhibitory action on NA-induced Ca²⁺ mobilization. Midazolam attenuates NA-induced Ca²⁺ influx via its inhibition of both nicardipine-sensitive and -insensitive pathways. Furthermore, midazolam attenuates the NA-induced release of Ca²⁺ from the storage sites. This effect contributes to the midazolam-induced inhibition of the NA-induced phasic contraction.

     

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.

     

Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca²⁺ ([Ca²⁺]_i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca²⁺]_i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized.
2. Histamine and substance P stimulated [³H]-inositol monophosphate ([³H]-IP₁) accumulation in U373 MG cells. Concentration-response curves of [³H]-IP₁ accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li⁺ yielded best-fit EC₅₀ values of 19.1±1.5 μM for histamine and 5.7±1.3 nM for substance P.
3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 μM histamine resulted in a transient 597±50 nM increase in [Ca²⁺]_i. The best-fit EC₅₀ for histamine was 4.6±2.2 μM. The initial, transient, histamine response was often followed by further small transient increases in [Ca²⁺]_i.
4. Treatment of U373 MG cells with 5 μM thapsigargin, followed by the readdition of 1.8 mM Ca²⁺ to the perfusion buffer, resulted in a steady-state level of [Ca²⁺]_i 97±5 nM above pretreated levels (measured 400 s after readdition of Ca²⁺). Perfusion of histamine (100 μM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca²⁺]_i. This effect of histamine was normally reversible upon washout. The best-fit EC₅₀ for the histamine response was 0.8±0.2 μM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca²⁺]_i.
5. Neither 100 μM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn²⁺ in U373 MG cells pretreated with 5 μM thapsigargin, indicating that the depressant effect on steady-state raised [Ca²⁺]_i was probably not due to a block of Ca²⁺ entry.
6. The depressant effect of histamine on [Ca²⁺]_i was blocked by 1 μM mepyramine, and was partially reduced by pre-incubation with 1 μM staurosporine (61±7% reduction) and with Ro 31-8220 (24±10% and 50±6% reduction by 1 and 10 μM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine.
7. Neither 1 μM staurosporine nor 10 μM KN-62 inhibited the binding of [³H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17±1% and 55±2% by 1 and 10 μM Ro 31-8220, respectively. However, [³H]-IP₁ accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 μM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 μM, similarly potentiated the response to 100 μM histamine in 3 out of 4 experiments. KN-62 (10 μM) did not stimulate histamine-induced [³H]-IP₁ accumulation.
8. In HEPES buffer to which no Ca²⁺ had been added, histamine stimulated a transient 451±107 nM increase in [Ca²⁺]_i. Pretreatment with 1 μM and 10 μM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca²⁺]_i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca²⁺]_i. H-89 did not alter the histamine response.
9. The effect of histamine in stimulating Ca²⁺ extrusion was not confined to U373 MG cells, since 100 μM histamine also caused a rapid decrease in steady-state levels of [Ca²⁺]_i in thapsigargin-treated human HeLa cells.
10. The results indicate that agonists which increase [Ca²⁺]_i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca²⁺]_i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca²⁺-extrusion pump.

     

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.

     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca²⁺ ([Ca²⁺]_i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca²⁺ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca²⁺ and an influx of extracellular Ca²⁺, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA₂ activation.
2. In RAW 264.7 cells UTP (100 μM) and thapsigargin (1  μM) caused 2 and 1.2 fold increases, respectively, in [³H]-AA release. The release of [³H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca²⁺-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA₂ inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml⁻¹) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA₂) had no effect on UTP and thapsigargin responses.
3. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (10 μM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca²⁺]_i rise.
4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM–3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release.
5. Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca²⁺]_i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA.
6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC.
7. Following treatment of cells with SK&F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca²⁺ influx, and the accompanying AA release, were down-regulated.
8. Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin.
9. We conclude that UTP-induced cPLA₂ activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca²⁺, which is essential for the activation of cPLA₂ by UTP and thapsigargin. The [Ca²⁺]_i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca²⁺]_i was also shown to be involved in AA release.