nM-1 microM Research Articles

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition.

Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

Endomorphin-1 and endomorphin-2 activate mu-opioid receptors in myenteric neurons of the guinea-pig small intestine.

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central mu-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM - 1 microM) inhibited (-log EC50 values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), caused a concentration-dependent (30 nM-1 microM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA2 values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective delta-receptor antagonist) or 30 nM nor-binaltorphimine (a selective kappa-receptor antagonist). These results demonstrate that endomorphins selectively activate mu-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists.

Desensitization of the histamine H1-receptor and transcriptional down-regulation of histamine H1-receptor gene expression in bovine tracheal smooth muscle.

We have investigated the role of protein kinase C (PKC) in the desensitization of histamine H1-receptors and in the expression of the histamine H1-receptor gene in airway smooth muscle. Prolonged 4beta-phorbol 12,13 dibutyrate (PDBu) pretreatment (4 h, 100 nM-1 microM) of bovine trachealis caused a concentration-dependent loss of contraction in response to histamine H1-receptor stimulation, which was associated with a concentration-dependent decrease in histamine-induced total [3H]-inositol phosphates accumulation. In contrast, the responses to sodium fluoride, a direct G-protein activator, were unalterd by PDBu (100-300 nM) pre-incubation and only slightly reduced following incubation with 1 microM PDBu. A selective PKC inhibitor, GF 109203X, partially blocked the PDBu (1 microM)-induced desensitization and completely blocked the effect of 100 nM PDBu, confirming the involvement of PKC. Binding experiments using [3H]-pyrilamine revealed a class of high-affinity binding sites within the range for the histamine H1 receptor in airway smooth muscle. PDBu (1 microM) pretreatment for 4 h did not change the number of histamine H1 receptors. PDBu (1 microM) exposure caused a time-dependent reduction in the steady-state levels of histamine H1-receptor mRNA, which was inhibited by pre-incubation with GF 109203X and by cycloheximide, a protein synthesis inhibitor. Nuclear run-on assays revealed a 50% reduction in the rate of histamine H1-receptor gene transcription after 17 h PDBu pretreatment, whereas mRNA stability was not affected by PDBu pretreatment (17 h). In conclusion, we have shown a PKC-mediated desensitization of the histamine H1-receptor in BTSM and a transcriptional down-regulation of the histamine H1-receptor gene expression, which requires new protein synthesis.

Exogenous and endogenous catecholamines inhibit the production of macrophage inflammatory protein (MIP) 1 alpha via a beta adrenoceptor mediated mechanism.

Noradrenaline (NA) and adrenaline (Ad) are modulators of cytokine production. Here we investigated the role of these neurotransmitters in the regulation of macrophage inflammatory protein (MIP)-1alpha expression. Pretreatment of RAW 264.7 macrophages with NA or Ad decreased, in a concentration-dependent manner (1 nM-100 microM), MIP-1alpha release induced by bacterial lipopolysaccharide (LPS 10 ng ml(-1) LPS). The effect of NA was reversed by the selective beta-adrenoceptor antagonist propranolol (10 microM), but not by the alpha-adrenoceptor antagonist phentolamine (10 microM). In the concentration range of 10 nM-10 microM, isoproterenol, a beta-adrenoceptor agonist, but not phenylephrine (a selective alpha1-adrenoceptor agonist) or UK-14304 (a selective alpha2-adrenoceptor agonist) mimicked the inhibitory effects of catecholamines on MIP-1alpha production. Increases in intracellular cyclic adenosine monophosphate, elicited either by the selective type IV phosphodiesterase inhibitor rolipram (0.1 - 10 microM), or by prostaglandin E2, (10 nM-10 microM) decreased MIP-1alpha release, suggesting that increased cyclic AMP may contribute to the suppression of MIP-1alpha release by beta-adrenoceptor stimulation. Northern blot analysis demonstrated that NA (100 nM-10 microM), Ad, isoproterenol, as well as rolipram (100 nM-10 microM) decreased LPS-induced MIP-1alpha mRNA accumulation. NA and Ad (1-100 microM) also decreased MIP-1alpha production in thioglycollate-elicited murine peritoneal macrophages. Pretreatment of mice with either isoproterenol (10 mg kg(-1), i.p.) or rolipram (25 mg kg(-1), i.p.) decreased LPS-induced plasma levels of MIP-1alpha, while propranolol (10 mg kg(-1), i.p.) augmented the production of this chemokine, confirming the role of a beta-adrenoceptor mediated endogenous catecholamine action in the regulation of MIP-1alpha production in vivo. Thus, based on our data we conclude that catecholamines are important endogenous regulators of MIP-1alpha expression in inflammation.

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 microM, reached a maximum of 45.3+/-2.8%, and was abolished by endothelial removal or treatment with L-NAME (100 microM), 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 microM) on endothelium-containing rings or adenosine (0.1 microM-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 microM-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.

Synaptic inhibitory effects of edrophonium on sympathetic ganglionic transmission.

To evaluate the effect of edrophonium on synaptic transmission in the superior cervical ganglion. In anaesthetized rats the effect of edrophonium on synaptic transmission was studied in vitro by testing whether it blocks the compound action potential recorded from postganglionic fibres evoked by stimulation of preganglionic axons. The superior cervical ganglion was excised and the cervical sympathetic trunk and internal carotid nerve were used for stimulating and recording, respectively. Drugs superfused included edrophonium (0.1-500 microM), neostigmine (0.1-10 microM), and muscarinic M1 and M2 antagonists pirenzepine and AFDX-116 (200 nM-10 microM), respectively. To evaluate a presynaptic action, the effect of edrophonium on basal and high-K+ (35 mM) evoked release of [3H]ACh from the superior cervical ganglion was studied in vitro. To evaluate a postsynaptic action, edrophonium's effect on postganglionic nerve discharge in response to arterial injection of ACh (100 micrograms) into the superior cervical ganglion was determined in vivo. Edrophonium (10-500 microM) decreased the compound action potential amplitude (ED50 163.5 microM). A decrease was not produced by neostigmine, nor was it reversed by pirenzepine or AFDX-116. Edrophonium blocked postganglionic cell firing in response to exogenously administered ACh. Although edrophonium did not affect basal or high-K+ evoked ACh release, when the evoked increase was calculated as a multiple of the basal release, it caused approximately a 30% (P < 0.005) reduction. Edrophonium blocks ganglionic cholinergic transmission postsynaptically and, possibly, presynaptically. The mechanism(s) by which this occurs does not appear to involve inhibition of cholinesterase, or activation of M1 or M2 receptor subtypes.

Modulation by substance P of synaptic transmission in the mouse hippocampal slice.

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra- or extracellular recording from the mouse hippocampal slice preparation. Bath-applied substance P (2-4 microM) or the selective NK1 receptor agonist substance P methylester (SPME, 10 nM-5 microM) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK1 receptors since it was completely blocked by the selective NK1 antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or 10 microM N-methyl-D-aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.

Dilator effect of bradykinin and acetylcholine in cerebral vessels after brain lesion

Vasodilation elicited by bradykinin (BK) or acetylcholine (Ach) (10 nM-10 microM) in isolated rat cerebral arteries was studied under control conditions, after sham treatment, and after cold lesion (placing a cooled metal probe on the exposed dura) of the cortex. After 24 or 48 hours, isometric force was measured in ring segments of basilar (BA) and middle cerebral arteries (MCA). Concentration-effect curves were constructed after precontraction with 100 microM uridine triphosphate (MCA) or 1 microM serotonin (BA). In MCA and BA, BK elicited similar relative relaxations with maxima of 40.9 +/- 1.5% and 40.7 +/- 3.1%, respectively, at 1 microM. Ach-induced relaxation in BA was much stronger with 82.0 +/- 5.8% at 1 microM. MCA did not relax consistently to Ach. Relaxation to BK in MCA segments was not different between sham-treated and untreated animals. After cold lesion, the dilation to BK (1 microM) was significantly reduced at 24 hours from 0.7 +/- 0.06 to 0.4 +/- 0.06 mN. At 48 hours, this decrease was partly reversed (to 0.5 +/- 0.07 mN). In BA, there was no difference in Ach-induced relaxation between cold-lesioned or sham-treated animals. In summary, the nitric oxide (NO)-mediated response to BK in MCA is attenuated 24 hours after cold lesion. This damage to the BK/NO system is partly reversed 48 hours after the lesion.

L-Type Calcium Channels Mediate a Slow Excitatory Synaptic Transmission in Rat Midbrain Dopaminergic Neurons

Patch pipettes were used to record whole-cell synaptic currents under voltage-clamp in dopaminergic neurons in slices of rat substantia nigra pars compacta and ventral tegmental area. We report that dihydropyridines (DHPs), L-type Ca2+ channel antagonists, depressed a slow EPSC (EPSCslow) evoked by a train of focally delivered electrical stimuli. In fact, the amplitude of the EPSCslow was reduced by the DHP antagonists nifedipine (1-100 microM), nimodipine (1-100 microM), and isradipine (30 nM-100 microM) in a concentration-dependent and reversible manner. On the other hand, Bay-K 8644 (1 microM), an L-type Ca2+ channel agonist, increased the EPSCslow. The DHPs depressed the EPSCslow only when the high-frequency stimulation that was used to evoke this synaptic current lasted >70 msec. On the other hand, Bay-K 8644 increased the amplitude of the EPSCslow only when it was evoked by a train <70 msec. Moreover, the DHPs did not affect the EPSCfast, the IPSCfast, and the IPSCslow. The inhibition of the EPSCslow caused by the DHPs is attributed to presynaptic mechanisms because (1) the inward current generated by exogenously administered glutamate was not affected and (2) the EPSCslow was reduced to a similar degree even when the activation state of postsynaptic L-type Ca2+ channels was changed by holding the neurons at -100, -60, and +30 mV. Finally, a DHP-sensitive component of the EPSCslow could even be detected after the blockade of N-, Q-, and P-type Ca2+ channels by the combination of omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. Taken together, these results indicate that under certain patterns of synaptic activity, L-type Ca2+ channels regulate the synaptic release of excitatory amino acids on the dopaminergic neurons of the ventral mesencephalon.

Inhibition of tumour necrosis factor and reversal of endotoxin-induced shock by U-83836E, a 'second generation' lazaroid in rats.

1. Antioxidants can exert protective effects in endotoxic shock by either a reduction of the oxidant damage or attenuation of Tumour Necrosis Factor (TNF-alpha) production. 2. Lazaroids are a family of compounds that inhibit lipid peroxidation. Besides, they can also reduce TNF-alpha. U-83836E is a new lazaroid lacking the glucocorticoid ring. 3. Aim of our study was to investigate the effect of U-83836E on TNF-alpha production either in vivo or in vitro. Endotoxic shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of S. enteritidis lipopolysaccharide (LPS). LPS administration reduced survival rate (0% survival, 72 h after endotoxin administration), decreased mean arterial blood pressure, increased serum and macrophage TNF-alpha and enhanced plasma malonylaldehyde (MAL) levels. Furthermore aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nM-10 microM). 4. Treatment with U-83836E (7.5, 15 and 30 mg kg(-1), i.v.) 5 min after endotoxin challenge significantly protected against LPS induced lethality (90% survival rate and 80% survival rate 24 h and 72 h after LPS injection respectively, following the highest dose of the drug), reduced hypotension, blunted plasma MAL, decreased serum and macrophage TNF-alpha and restored the hyporeactivity of aortic rings to control values. In vitro LPS stimulation (50 microg ml(-1) for 4 h) significantly increased cytokine production in macrophages (Mphi) harvested from untreated normal rats. Pretreatment with pertussis toxin (PT; 0.1, 1 and 10 ng ml(-1) 4 h before LPS) significantly increased TNF-alpha production. PT effects on these LPS responses were correlated with a PT mediated ADP ribosylation of a 41 kDa protein. U-83836E (50 microM) reduced, in a dose dependent manner, LPS induced TNF-alpha production and inhibited the PT effects on cytokine production and on ADP ribosylation of the protein. 5. Our data suggest that lazaroids may affect the early events associated with LPS receptor mediated activation of a G protein in LPS induced TNF-alpha production. These molecular events may explain, at least in part, the in vivo inhibition of cytokine production and reversal of endotoxic shock.

Spatial heterogeneity in the mechanisms contributing to acetylcholine-induced dilatation in the rabbit isolated ear.

1. Using an X-ray microangiographic technique in rabbit isolated perfused ears preconstricted with 5-HT (300 nM) and histamine (300 nM), we investigated the combined actions of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin on acetylcholine-induced depressor responses. 2. Under control conditions, acetylcholine (10 nM-30 microM) induced a concentration-dependent reversal of the pressor response, reaching a maximum of 66.0+/-13.6% (n = 6). In the presence of L-NAME (300 microM) and indomethacin (10 microM), this depressor action was reduced, reaching a maximum of 38.6+/-5.9% (n = 6). 3. The control response was associated with substantial vasodilatation in the central ear artery (G0), a smaller dilatory action on first generation branch arteries (G1) and no effect on second generation branch arteries (G2). In the presence of L-NAME and indomethacin, vasodilatation occurred in G2 with no effect in G0 or G1. 4. Two calcium-activated K+ channels blockers, charybdotoxin (ChTX; 10 nM) and penitrem A (100 nM), further inhibited, but did not abolish, the L-NAME- and indomethacin-resistant response to acetylcholine (10 nM-300 microM). Both agents abolished the vasodilatory action of acetylcholine in G2. 5. In conclusion, L-NAME and indomethacin induced a shift in acetylcholine-induced vasodilatation from G0 and G1 to G2. This is consistent with the suggestion that nitric oxide dominates in larger vessels whilst other mechanisms dominate in smaller vessels. The L-NAME- and indomethacin-resistant component was inhibited by ChTX and penitrem A, suggesting it is mediated, at least in part, by activation of K(Ca) channels and could therefore involve a hyperpolarising mediator such as endothelium-derived hyperpolarising factor.

Characterization of prostanoid receptor-evoked responses in rat sensory neurones.

1. Prostanoid receptor-mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F-11 (embryonic rat DRG x neuroblastoma hybrid) cell line. 2. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2alpha had negligible effects. The rank order of agonist potency was cicaprost>PGE2=BMY45778=cPGI2=PGI2. In the F-11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2++ +. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. 3. To examine the effects of prostanoids on C-fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the 'grease-gap' technique. PGI2 (0.1 nM-10 microM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2alpha. 4. Prior depolarization of nerves with either forskolin (10 microM) or phorbol dibutyrate (1 microM) alone significantly reduced the response to PGI2 (10 microM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. 5. Putative EP1 and/or TP receptor-selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. 6. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.

Differential effects of somatostatin and angiopeptin on cell proliferation.

1. Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst2/sst5 receptor-selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO-K1 cells stably transfected with the human or rat recombinant sst2 or sst5 receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors. 2. In CHO-KI cells, expressing either human or rat recombinant sst2 or sst5 receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1-1000 nM) had no effect on basal re-growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml(-1)) stimulated re-growth of these cells. 3. SRIF (0.1-1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated re-growth in CHO-K1 cells expressing human sst2 (h sst2) or sst5 (h sst5) receptors (pIC50=8.05+/-0.03 and 8.56+/-0.12, respectively). In contrast, angiopeptin (0.1-1000 nM) acted as a partial agonist at the h sst2 receptor (44.6+/-2.7% inhibition of the bFGF-stimulated re-growth at 100 nM; pIC50=8.69+/-0.25) but was devoid of any agonist activity at the h sst5 receptor. 4. In CHO-K1 cells stably expressing rat recombinant sst2 (r sst2) or sst5 (r sst5) receptors, SRIF (0.1-1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC50=7.98+/-24 and 8.50+/-0.12, respectively). Angiopeptin (0.1-1000 nM) caused a concentration-dependent inhibition of bFGF-stimulated re-growth at the r sst2 receptor (pIC50=8.08+/-0.24) but acted as a partial agonist at the r sst5 receptor (maximum response= 57.7+/-3.6% inhibition of bFGF-stimulated re-growth at 100 nM; pIC50=8.60+/-0.16). 5. Although angiopeptin was inactive as an agonist at the h sst5 receptor, 100 nM angiopeptin potently antagonized the SRIF-induced inhibition of proliferation in CHO h sst5 (estimated pKB= 10.4+/-0.3). 5-Hydroxytryptamine (0.1 nM-10 microM) also inhibited bFGF-stimulated re-growth (pIC50=8.36+/-0.11) and angiopeptin had no effect on this response (pKB<7). 6. SRIF (0.1-1000 nM) caused a concentration-dependent (pIC50=8.04+/-0.08) inhibition of bFGF-stimulated re-growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF-stimulated re-growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration-effect curve to SRIF with an estimated pKB value of 7.70+/-0.12. 7. These findings suggest that the low intrinsic activity of angiopeptin at the h sst2 receptor, combined with its lack of agonist activity at the h sst5 receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

Modulation of Ca2+ channel currents of acutely dissociated rat periaqueductal grey neurons.

1. The actions of the neuropeptide nociceptin on the calcium channel currents (IBa) of acutely dissociated rat periaqueductal grey (PAG) neurons were examined using whole-cell patch clamp techniques. These effects were compared with those of opioid receptor agonists and the GABAB receptor agonist baclofen. 2. Neurons from young adult rats (23 to 56 days old) expressed predominantly omega-conotoxin GVIA (N-type)- and omega-agatoxin IVA (P/Q-type)-sensitive IBa, together with smaller amounts of nimodipine-sensitive current and current resistant to all three blockers. There was proportionately more N-type IBa in neurons from female rats and proportionately more resistant current in neurons from male rats. 3. Nociceptin (EC50, 5 nM) and baclofen (EC50, 0.8 microM) inhibited IBa in all PAG neurons, while the opioid agonist methionine enkephalin (met-enkephalin; 300 nM-10 microM) inhibited IBa in 40 % of neurons. The effects of met-enkephalin were reversed by the mu-opioid antagonist CTAP, and mimicked by the mu-opioid agonist DAMGO (300 nM-3 microM). The delta-opioid agonists DPDPE and deltorphin II, and the kappa-opioid agonist U69593, did not affect IBa in any neuron. The actions of nociceptin were not mimicked or blocked by the opioid antagonist naloxone or the nociceptin analogue [desPhe1]-nociceptin. 4. The effects of nociceptin and baclofen on IBa were blocked by pretreatment of the neurons with pertussis toxin (500 ng ml-1, 8 h). 5. Nociceptin predominantly inhibited the N-type (EC50, 2 nM; maximum inhibition, 50 %) and P/Q-type (EC50, 7 nM; maximum inhibition, 33 %) IBa while having little effect on the L-type and R-type IBa. 6. These results are consistent with the previously described actions of nociceptin, baclofen and micro-opioids in PAG slices, whereby they couple to increases in an inwardly rectifying K+ conductance. These agonists thus have the potential to modulate the function of PAG neurons via a number of different cellular effectors.

Galanin inhibits glucagon-like peptide-1 secretion through pertussis toxin-sensitive G protein and ATP-dependent potassium channels in rat ileal L-cells.

The neuropeptide galanin is widely distributed in the gastrointestinal tract and exerts several inhibitory effects, especially on intestinal motility and on insulin release from pancreatic beta-cells. The presence of galanin fibres not only in the myenteric and submucosal plexus but also in the mucosa, prompted us to investigate the regulatory role of galanin, and its mechanism of action, on the secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). Rat ileal cells were dispersed through mechanical vibration followed by moderate exposure to hyaluronidase, DNase I and EDTA, and enriched for L-cells by counterflow elutriation. A 6- to 7-fold enrichment in GLP-1 cell content was registered after elutriation, as compared with the crude cell preparation (929 +/- 81 vs 138 +/- 14 fmol/10(6) cells). L-cells then accounted for 4-5% of the total cell population. Bombesin induced a time-(15-240 min) and dose- (0.1 nM-1 microM) dependent release of GLP-1. Glucose-dependent insulinotropic peptide (GIP, 100 nM), forskolin (10 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA, 1 microM) each stimulated GLP-1 secretion over a 1-h incubation period. Galanin (0.01-100 nM) induced a dose-dependent inhibition of bombesin- and of GIP-stimulated GLP-1 release (mean inhibition of 90% with 100 nM galanin). Galanin also dose-dependently inhibited forskolin-induced GLP-1 secretion (74% of inhibition with 100 nM galanin), but not TPA-stimulated hormone release. Pretreatment of cells with 200 ng/ml pertussis toxin for 3 h, or incubation with the ATP-sensitive K+ channel blocker disopyramide (200 microM), prevented the inhibition by galanin of bombesin- and GIP-stimulated GLP-1 secretion. These studies indicate that intestinal secretion of GLP-1 is negatively controlled by galanin, that acts through receptors coupled to pertussis toxin-sensitive G protein and involves ATP-dependent K+ channels.

Neuroregulation of mucus secretion by opioid receptors and K(ATP) and BK(Ca) channels in ferret trachea in vitro.

1. Opioid agonists inhibit neurogenic mucus secretion in the airways. The mechanism of the inhibition is unknown but may be via opening of potassium (K+) channels. We studied the effect on neurogenic secretion in ferret trachea in vitro of the OP1 receptor (formerly known as delta opioid receptor) agonist [D-Pen2,5]enkephalin (DPDPE), the OP2 receptor (formely kappa) agonist U-50,488H, the OP3 receptor (formerly micro) agonist [D-Ala2, N-Me-Phe, Gly-ol5]enkephalin (DAMGO), the ATP-sensitive K+ (K(ATP)) channel inhibitor glibenclamide, the large conductance calcium activated K+ (BK(Ca)) channel blocker iberiotoxin, the small conductance K(Ca) (SK(Ca)) channel blocker apamin, the K(ATP) channel opener levcromakalim, a putative K(ATP) channel opener RS 91309, and the BK(Ca) channel opener NS 1619. Secretion was quantified by use of 35SO4 as a mucus marker. 2. Electrical stimulation increased tracheal secretion by up to 40 fold above sham-stimulated levels. DAMGO or DPDPE (10 microm each) significantly inhibited neurogenic secretion by 85% and 77%, respectively, effects which were reversed by naloxone. U-50,488H had no significant inhibitory effect on neurogenic secretion, and none of the opioids had any effect on ACh-induced or [Sar9]substance P-induced secretion. 3. Inhibition of neurogenic secretion by DAMGO or DPDPE was reversed by iberiotoxin (3 microM) but not by either glibenclamide or apamin (0.1 microM each). Iberiotoxin alone did not affect the neurogenic secretory response. 4. Levcromakalim, RS 91309 or NS 1619 (3 nM-3 microM) inhibited neurogenic secretion with maximal inhibitions at 3 microM of 68%, 72% and 96%, respectively. Neither levcromakalim nor RS 91309 at any concentration tested significantly inhibited acetylcholine (ACh)-induced secretion, whereas inhibition (60%) was achieved at the highest concentration of NS 1619, a response which was blocked by iberiotoxin. 5. Inhibition of neurogenic secretion by levcromakalim (3 microM) or RS 91309 (30 nM) was inhibited by glibenclamide but not by iberiotoxin. In contrast, inhibition by NS 1619 (30 nM and 3 microM) was blocked by iberiotoxin but not by glibenclamide. 6. We conclude that, in ferret trachea in vitro, OP1 or OP3 opioid receptors inhibit neurogenic mucus secretion at a prejunctional site and that the mechanism of the inhibition is via opening of BK(Ca) channels. Direct opening of BK(Ca) channels or K(ATP) channels also inhibits neurogenic mucus secretion. In addition, opening of BK(Ca) channels inhibits ACh-evoked secretion of mucus. Drugs which open BK(Ca) channels may have therapeutic anti-secretory activity in bronchial diseases in which neurogenic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro.

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea. 2. The selective 5-HT2 receptor antagonist ketanserin (0.1 microM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA). 3. The partial agonist for 5-HT1A receptors, 8-OH-DPAT (1 microM), and the full agonist for 5-HT1 receptors, 5-CT (0.3 microM), potentiated the submaximal contractions induced by the 5-HT2 receptor agonist alpha-methyl-5-HT (0.1 microM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 microM), as well as the 5-HT1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 microM each), caused significant inhibition of the tracheal contractions induced both by NKA (10 nM-3 microM) and 5-HT (10 nM-10 microM) (n=4-10). This suggests that activation of 5-HT1A receptors potentiates the 5-HT2 receptor-mediated contractions. 4. SDZ 216525 (0.1 microM) significantly reduced the maximal contraction produced by 1 microM NKA (n=10, P< 0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells. 5. Even in the presence of atropine (1 microM), 8-OH-DPAT (1 microM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 microM), SDZ 216525 (0.1 microM), NAN-190 (0.1 microM) and 8-OH-DPAT (1 microM) (n=4-6). These data demonstrate that the 5-HT1A receptor-mediated potentiation of contractile responses is not due to nonspecific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation. 6. The selective 5-HT1B/1D receptor antagonist GR 127935, the selective 5-HT3 receptor antagonist tropisetron and the selective 5-HT4 receptor antagonists SB 204070 and GR 113808 (0.1 microM each) had no effect on the concentration-response curve for NKA (n=6-10), ruling out the involvement of 5-HT1B/1D, 5-HT3 and 5-HT4 receptors. 7. The alpha-adrenoreceptor antagonist phentolamine (1 microM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of alpha-adrenoreceptors. 8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT2 receptors. Activation of 5-HT1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT2 receptor activation. The 5-HT1B/1D, 5-HT3 and 5-HT4 receptors are not involved in the NKA-induced contraction of rat airways.

Effects of bradykinin on signal transduction, cell proliferation, and cytokine, prostaglandin E2 and collagenase-1 release from human corneal epithelial cells.

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B2-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells. 2. The aims of the present studies were to demonstrate the specific binding of [3H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca2+-mobilization ([Ca2+]i), cell proliferation (via [3H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E2 (PGE2). 3. Specific [3H]-BK binding comprised 83 +/- 2% of the total binding, and was of high affinity (Kd = 1.66 +/- 0.52 nM, n = 5), saturable (Bmax = 640 +/- 154 fmol g(-1) wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]BK; icatibant): Ki = 0.17 +/- 0.07 nM; BK: Ki = 1.0 +/- 0.11 nM; [Tyr8]-BK: Ki = 12.9 +/- 2.3 nM; [des-Arg9]-BK: Ki > 9,200 nM (all n = 3-5)). 4. BK potently stimulated PI turnover (EC50 = 2.3 +/- 0.3 nM; n = 7) and [Ca2+]i mobilization (EC50 = 8-20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM-10 microM Hoe-140, a selective B2-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione) (IC50 = 3.0 +/- 1.6 microM). BK-induced [Ca2+]i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine. 5. BK (0.1 nM-10 microM) significantly (P<0.05-0.001) stimulated [3H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1alpha (IL-1alpha; 10 ng ml(-1)) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM-10 microM) was without effect. 6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 microg ml(-1)) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE2 from CEPI cells, BK (0.1 nM-10 microM) was without any significant effect under these conditions. 7. In conclusion, these data indicate that the CEPI cells express high-affinity [3H]-BK binding sites representing B2-subtype BK receptors coupled to PI turnover and [Ca2+]i mobilization which appear to stimulate [3H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE2, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

Effect of 17beta-oestradiol on cytokine-induced nitric oxide production in rat isolated aorta.

1. Studies were performed on isolated aortic rings without endothelium to investigate the effect of 17beta-oestradiol on cytokine-induced nitric oxide production by the inducible nitric oxide synthase (iNOS). 2. Treatment of the isolated aortic rings with interleukin-1beta (IL-1beta, 20 micro ml(-1)) led to the expression of iNOS mRNA and protein, as well as significant nitrite accumulation in the incubation media and suppression of phenylephrine (1 nM-10 microM)-evoked contraction. 3. Cycloheximide (1 microM), a protein synthesis inhibitor, prevented iNOS protein expression, nitrite accumulation and the suppression of contractility by IL-1beta on the isolated aortic rings. 17Beta-oestradiol (1 nM-10 microM) and the partial oestrogen receptor agonist 4-OH-tamoxifen (1 nM-10 microM) produced concentration-dependent inhibition of IL-1beta-induced nitrite accumulation and restored vasoconstrictor responsiveness to phenylephrine, similar to the iNOS inhibitor aminoguanidine (100 microM). 4. Semiquantitative PCR demonstrated decreased iNOS mRNA in the IL-1beta-induced and 17beta-oestradiol-treated rings. Western blot analysis of rat aorta homogenates revealed that 17beta-oestradiol treatment resulted in a reduction in IL-1beta-induced iNOS protein level. 5. Incubation with tumour necrosis factor alpha (TNF alpha, 1 ng ml(-1)) resulted in significant nitrite accumulation in the incubation media and suppression of the smooth muscle contractile response to phenylephrine, similar to IL-1beta. The effects of TNF alpha were also inhibited by co-incubation of the rings with 17beta-oestradiol and 4-OH-tamoxifen (1 microM). 6. The anti-transforming growth factor-beta1 (TGF-beta1) antibody, which inhibited TGF-beta1-induced suppression of nitrite production from IL-1beta-treated vascular rings, did not affect the inhibitory action of 17beta-oestradiol, suggesting that the effect of oestrogen on iNOS inhibition was not mediated by TGF-beta1. 7. These results show that the ovarian sex steroid, 17beta-oestradiol is a modulator of cytokine-induced iNOS activity in rat vascular smooth muscle and its mechanism of action involves decrease of iNOS mRNA and protein.

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype.

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.