Common Site Of Action Research Articles

Serum Fibroblast Growth Factor 21 Levels in Children and Adolescents with Hashimoto’s Thyroiditis before and after l-Thyroxin Medication: A Prospective Study

Backgrounds and Objectives: Fibroblast growth factor 21 (FGF-21) is a complex hormone, sharing common sites of action with thyroid hormones. We investigated the association among FGF-21 levels, resting metabolic rate (RMR), and l-thyroxin (LT4) treatment in children and adolescents with Hashimoto’s thyroiditis. Materials and Methods: A total of 60 youngsters with chronic autoimmune thyroiditis (AIT) (30 with subclinical hypothyroidism, 30 with euthyroidism) and 30 age and sex-matched healthy participants (5–18 years old) were enrolled in the study. Anthropometric, biochemical parameters, and RMR levels were assessed in all participants; serum FGF-21 levels were measured in the control group and the group with subclinical hypothyroidism before and six months after medication with LT4. Results: FGF-21 levels were lower in the treatment group compared with the healthy ones, but this difference was not statistically significant (p > 0.05); despite the increase in FGF-21 levels after six months of LT4 treatment, this difference was not statistically significant (p > 0.05). Free thyroxin (FT4) levels correlated well with FGF-21 levels (r = 0.399, p < 0.01), but further analysis revealed no interaction between these two variables. Both patient groups presented elevated triglyceride (TG) levels compared to controls (p < 0.05). LT4 treatment had no impact on RMR and lipid or liver or glycaemic parameters. An increase in fat mass and fat-free mass were reported, independently of FGF-21 levels. Conclusions: In youngsters with subclinical hypothyroidism due to Hashimoto’s thyroiditis, the serum FGF-21 levels are not significantly lower than in healthy individuals and increase after treatment with LT4 without a statistical significance. Further studies with a large number of young patients and severe hypothyroidism are recommended to confirm our results.

Non-invasive brain stimulation (rTMS and tDCS) in patients with aphasia: Mode of action at the cellular level

A high proportion of patients who have suffered a stroke also suffer from aphasia. Approximately half of those affected will remain in this state despite intensive language therapy. Non-invasive brain stimulation allows us to directly and focally stimulate areas of the brain. Repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS), methods used in the treatment of aphasia, are based on an imbalance of mutual interhemispheric inhibition. In open and sham-controlled studies, a low-frequency, 1Hz stimulation of the non-lesioned hemisphere (the homologue of Broca's area) for a week or more significantly improved spontaneous speech and anomia in patients with non-fluent aphasia. These positive outcomes from rTMS stimulation developed slowly, often over months following treatment, and persisted. Effects of intermittent theta burst stimulation (iTBS) developed faster than the low-frequency stimulation, and high-activity enhancement was detected in the left hemisphere after the stimulation of Broca's region. Both types of tDCS stimulation resulted in improved comprehension and reduced anomia, their primary modes of action are distinct, however, both share a common site of action with regard to the balance that occurs between inhibitory and excitatory neurotransmitters (synaptic and non-synaptic). Both types of non-invasive stimulation prepare the lesioned brain for better outcome.

Increase of paradoxical excitement response during propofol-induced sedation in hazardous and harmful alcohol drinkers

Paradoxical excitement response during sedation consists of loss of affective control and abnormal movements. Chronic alcohol abuse has been proposed as a predisposing factor despite lack of supporting evidence. Because alcohol and propofol have a common site of action, we postulated that paradoxical excitement responses during propofol-induced sedation occur more frequently in hazardous and harmful alcohol drinkers than in social or non-drinkers. One hundred and ninety patients undergoing orthopaedic knee joint surgery were enrolled in this prospective and observational study. Subjects were divided into Group HD (hazardous and harmful drinkers) or Group NHD (no hazardous drinkers) according to the alcohol use disorder identification test (AUDIT). In study 1, propofol infusion was adjusted to achieve the bispectral index at 70-80 using target-controlled infusion. In study 2, the target concentration of propofol was fixed at 0.8 (study 2/Low) or 1.4 μg ml(-1) (study 2/High). Paradoxical excitement responses were categorized by intensity into mild, moderate, or severe. The overall incidence of paradoxical excitement response was higher in Group HD than in Group NHD in study 1 (71.4% vs 43.8%; P=0.022) and study 2/High (70.0% vs 34.5%; P=0.006) but not in study 2/Low. The incidence of moderate-to-severe response was significantly higher in Group HD of study 1 (28.6% vs 3.1%; P=0.0005) and study 2/High (23.3% vs 3.4%; P=0.029) with no difference in study 2/Low. Severe excitement response occurred only in Group HD of study 1 and study 2/High. Paradoxical excitement occurred more frequently and severely in hazardous and harmful alcohol drinkers than in social drinkers during propofol-induced moderate-to-deep sedation, but not during light sedation.

The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase

Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

Conformational analysis of convulsant and anticonvulsant barbiturates

In an effort to obtain definitive structure-activity relationships for the convulsant and anticonvulsant barbiturates a theoretical and experimental study was undertaken of the molecular conformations of 16 optical, geometric, and structural isomers of pentobarbitone and its homologues, four of which are convulsant. Initially classical calculations were used to obtain approximate potential energy surfaces, including quantitative estimates of barriers and the locations of energy minima. The latter conformations were further refined by minimizing total energy with respect to all geometric variables using mindo/3. Quantitative agreement between the classical and quantum mechanical calculations was generally good. The rotational barriers between low energy conformations were higher than expected, suggesting that barbiturates may be conformationally restricted even at physiological temperatures. This was confirmed by 1H and 13C nuclear magnetic resonance. In each case the observed solution conformations fell within minimum energy regions on the calculated potential energy surfaces. For the purpose of correlating conformation with biological activity we did not assume that the lowest energy gas phase or solution conformations were active, but included all energy minima within 10 kcal/mole of the global minimum. Only two conformations were thermally accessible to all the convulsants studied. Of these, the lower energy conformation bears a close structural resemblance, in key regions, to the potent natural convulsant picrotoxinin, with which the convulsant barbiturates may therefore share a common site of action.

Interaction of the nicotinic cholinergic system with ethanol withdrawal.

The observation that alcohol and nicotine are commonly abused together suggests that the two drugs have common sites of action. In vitro studies indicate that nicotinic acetylcholine receptor (nAChR) function is enhanced by ethanol. Furthermore, some ethanol-related behaviors are associated with a region of mouse chromosome 2 that contains the gene encoding the alpha4 subunit of the nAChR (Chrna4). We have identified a polymorphism in Chrna4 that results in an alanine (A) or threonine (T) residue at position 529 in the second intracellular loop of the protein. Nicotinic receptors expressing the A variant have greater responses to nicotine and ethanol than receptors with the T variant when measured in vitro, but the possible effects of the polymorphism on the severity of ethanol withdrawal have not been assessed. The handling-induced convulsion (HIC) assay is an established method for studying drug withdrawal in vivo. We monitored the HIC responses of mice for 8 h after an injection of ethanol (4 g/kg). A survey of 16 mouse strains, as well as previously published data, indicated an association of the A/T polymorphism with ethanol withdrawal. This association was also found in wild-type animals from an F2 intercross of the A/J (A529-genotype) strain with C57BL/6J (T529-genotype) mice that also lack expression of the beta2 nAChR subunit. Beta2 -/- animals, which do not express alpha4beta2 nAChRs in the brain, exhibited significantly lower HIC responses and no effect of the polymorphism. These results suggest that the nicotinic cholinergic system and the A/T polymorphism modulate ethanol withdrawal.

The effects of nandrolone decanoate on cocaine-induced kindling in male rats

Use and abuse of various controlled substances in recent years has reached alarming levels. Among these are cocaine and anabolic steroids. The two contrasting types of drug have common sites of action within the limbic system of the central nervous system. The ability of cocaine to provoke seizures is well documented, and sex hormones also have been shown to alter seizure types and characteristics. This project studied the consequences of co-administration of cocaine and a representative anabolic–androgenic steroid, nandrolone decanoate. Specifically, the effects of nandrolone on cocaine-induced kindling of seizures were examined. Nandrolone was shown to increase seizure rate when given in high (20 mg twice weekly) intermittent doses. No statistically significant differences were observed with low (2 mg) daily doses of nandrolone. The results support the hypothesis that an androgen may interact so as to modify the pattern of cocaine-related kindling. However, the potential of either pharmacodynamic and/or pharmacokinetic mechanism(s) for this interaction exists, and the nature of these interactions remains to be fully elucidated.

Ca2+, Sr2+, and Ba2+ Identify Distinct Regulatory Sites on Adenylyl Cyclase (AC) Types VI and VIII and Consolidate the Apposition of Capacitative Cation Entry Channels and Ca2+-sensitive ACs

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Effects of the combination of halothane and serotonin uptake blockers on synaptic transmission in the rat dentate gyrus in vitro.

Although the exact basis of their action remains unknown, volatile agents affect noradrenergic and serotoninergic systems. Imipramine and fluoxetine have documented effects on these neurotransmitter transmission systems. Given the common sites of action of these antidepressants and halothane, we examined their individual and combined effects on tonic excitatory post-synaptic potentials (EPSPs) and frequency dependent blockade in the rat dentate gyrus in vitro. Extracellular recordings of field EPSPs were maintained from the dentate gyrus, in the presence of picrotoxin (100 microM). Stimulation at 30 Hz (200 ms) allowed investigation of frequency dependent blockade. Once a stable equilibrium was established, halothane, imipramine and fluoxetine were administered via the perfusate and recordings were made. Halothane produced a dose dependent reduction in EPSP amplitude (EC50 0.28 mM; n = 12). Imipramine (1-10 microM) potentiated the EPSP amplitude (148.2 +/- 8.2%; imipramine 1 microM; n = 6). Fluoxetine (0.5-10 microM) reduced EPSP amplitude to 83.7 +/- 22.1% of control (n = 6). In the presence of halothane 0.2 mM, imipramine reduced the EPSP amplitude to 56.5 +/- 9.9% of control (imipramine 10 microM; n = 6; p < 0.05 compared with imipramine alone). Halothane (0.2 mM) demonstrated frequency dependent blockade. However, neither imipramine nor fluoxetine showed use dependent inhibition at the doses investigated. When combined with halothane 0.2 mM, fluoxetine 10 microM demonstrated frequency dependent blockade at the sixth pulse in the train compared with controls (13.8 +/- 4.7% vs 38.1 +/- 8.3%; n = 6; p < 0.05). The halothane-imipramine combination did not exhibit use dependent blockade greater than controls. The reversal of imipramine-induced EPSP potentiation by the preapplication of halothane has not been previously reported. It may be due to modulation of noradrenergic transmission by halothane. The frequency dependent blockade produced by the combination of fluoxetine 10 microM and halothane may be mediated by a nonspecific membrane effect on 5-HT uptake. These differing effects underline the broad action of volatile agents on synaptic mechanisms.

Involvement of Nitric Oxide in Parasympathetic and Antidromic Vasodilatations in Cat Lower Lip

The involvement of nitric oxide (NO) in the lower lip vasodilatations mediated via parasympathetic and antidromic mechanisms was examined in alpha-chloralose/urethane-anesthetized cats, with the two types of blood flow responses being recorded separately (by laser Doppler flowmeter) from the two sides of the lower lip. The central cut end of the lingual nerve (LN) or the peripheral cut end of the inferior alveolar nerve (IAN) was electrically stimulated to elicit parasympathetic or antidromic vasodilatation, respectively, in the lower lip. N(G)-nitro-L-arginine methyl ester (L-NAME), but not N(G)-nitro-D-arginine methyl ester (D-NAME) (each at 30 mg/kg), markedly reduced the increases in lip blood flow evoked by stimulation, the reduction being to a similar degree irrespective of whether LN or IAN was stimulated. Pretreatment with L-arginine did not prevent the L-NAME-induced attenuation of either type of vasodilatation. In conclusion, these results suggest that synthesized NO may have a common site of action in antidromic and parasympathetic vasodilator pathways to the cat lower lip.

Serotonin and Hallucinogens

This brief review traces the serotonin (5-HT) hypothesis of the action of hallucinogenic drugs from the early 1950s to the present day. There is now converging evidence from biochemical, electrophysiological, and behavioral studies that the two major classes of psychedelic hallucinogens, the indoleamines (e.g., LSD) and the phenethylamines (e.g., mescaline), have a common site of action as partial agonists at 5-HT2A and other 5-HT2 receptors in the central nervous system. The noradrenergic locus coeruleus and the cerebral cortex are among the regions where hallucinogens have prominent effects through their actions upon a 5-HT2A receptors. Recently, we have observed a novel effect of hallucinogens—a 5-HT2A receptor-mediated enhancement of nonsynchronous, late components of glutamatergic excitatory postsynaptic potentials at apical dendrites of layer V cortical pyramidal cells. We propose that an effect of hallucinogens upon glutamatergic transmission in the cerebral cortex may be responsible for the higher-level cognitive, perceptual, and affective distortions produced by these drugs.

Fencamfamine modulates sodium, potassium-ATPase through cyclic AMP and cyclic AMP-dependent protein kinase in rat striatum.

Dopamine (DA) and fencamfamine (FCF) modulatory action on Na,K-ATPase and Mg-ATPase activity were evaluated in rat striatum. DA and FCF induced a decrease in Na,K-ATPase, without affecting Mg-ATPase activity. The effect of FCF was dose-dependent from 10 to 100 microM, with an IC50 of 4.7 x 10(-5) M. Furthermore, the effect of FCF (100 microM) increasing AMPc levels, but not GMPc, was nonadditive with that of DA (10 microM), which is consistent to a common site of action. The 8-bromo-cyclic AMP also induced a specific reduction in the Na,K-ATPase activity. The reduction of Na,K-ATPase induced by FCF (100 microM) was blocked by either SCH 23390 or sulpiride, which are D1 and D2 receptor antagonists. The decrease in striatal NA,K-ATPase activity induced by FCF was blocked by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), but not by KT 5823, a selective inhibitor of cyclic GMP-dependent protein kinase (PKG). Otherwise, KT 5720 or KT 5823 did not produce any change in Na,K-ATPase or Mg-ATPase activity. These data suggest that FCF reduces Na,K-ATPase activity through cyclic AMP-dependent changes in protein phosphorylation via a PKA mechanism.

Halothane attenuates nitric oxide relaxation of rat aortas by competition for the nitric oxide receptor site on soluble guanylyl cyclase

Endothelial cells play an important role in the regulation of vascular activity through the release of endothelium derived relaxing factor (EDRF) now believed to be nitric oxide (NO). NO and the NO donor drug nitroglycerin relax vascular smooth muscle by stimulating soluble guanylyl cyclase leading to elevation of intracellular levels of cyclic guanosine 3′,5′-monophosphate (cGMP). Halothane has been shown to inhibit the action of NO on blood vessels. This study was designed to further investigate the mechanisms by which halothane attenuates NO-induced vascular relaxations. This was done by examining the effects of halothane on nitroglycerin and NO-induced relaxations in the presence and absence of the inhibitors of soluble guanylyl cyclase, methylene blue and 6-anilino-5,8-quinolinedione (LY 83583). Thoracic aortas from anesthetized male Sprague–Dawley rats were excised and cut into rings and the endothelium was removed. The aortic rings were suspended in organ baths containing Krebs solution and equilibrated at their optimal passive tension. When a stable plateau of contraction was produced by EC 60 concentrations of norepinephrine, increasing concentrations of nitroglycerin or NO were added to the baths to relax the rings. This contraction–relaxation procedure was repeated three or four times. In some baths halothane was administered by a calibrated vaporizer 10 min before beginning the second procedure. Either methylene blue or LY 83583 was added to the baths 20 min before the third procedure. The combination of halothane, methylene blue or LY 83583 was added before the fourth procedure. Halothane, methylene blue or LY 83583 significantly inhibited nitroglycerin-induced relaxation individually. Halothane and LY 83583 also significantly inhibited NO-induced relaxations (5×10 −9–3×10 −8 M and 5×10 −9–3×10 −5 M, respectively) individually. The combination of halothane and methylene blue or halothane and LY 83583 significantly inhibited nitroglycerin-induced relaxation, also, the combination of halothane and LY 83583 significantly inhibited NO-induced relaxations. Halothane, methylene blue and LY 83583 treatment led to rightward shift in the concentration–effect curves. Halothane, in combination with methylene blue or LY 83583, produced inhibition equivalent to the sum of their individual effects. The present study demonstrates that the halothane, methylene blue and LY 83583 attenuate nitroglycerin and NO-induced relaxations of endothelium-denuded rat aortic rings. This suggests that halothane, methylene blue and LY 83583 may act through competitive antagonism at a common site of action on soluble guanylyl cyclase in the EDRF/NO relaxation pathway.

Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor.

1. The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 micrograms kg-1, i.v.). 2. One-minute intracarotid (i.c.) infusions of 5-HT (0.3-30 micrograms min-1), 5-carboxamidotryptamine (5-CT; 0.01-0.3 microgram min-1), 5-methoxytryptamine (1-100 micrograms min-1) and lisuride (3-1000 micrograms min-1) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT > > 5-HT > or = 5-methoxytryptamine > lisuride, whereas cisapride (100-1000 micrograms min-1, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85 +/- 7 micrograms kg-1, i.c.), but not cisapride (mean dose of 67 +/- 7 micrograms kg-1, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (3-3000 micrograms min-1) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200 +/- 33 micrograms kg-1, i.c.) the agonist-induced vasodilator responses. 3. The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (+/-)-pindolol (4 mg kg-1) or ritanserin (100 micrograms kg-1) plus granisetron (300 micrograms kg-1), but were dose-dependently blocked by i.v. administration of methiothepin (10 and 30 micrograms kg-1, given after ritanserin plus granisetron), mesulergine (10 and 30 micrograms kg-1), metergoline (1 and 3 mg kg-1), methysergide (1 and 3 mg kg-1) or clozapine (0.3 and 1 mg kg-1). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine. 4. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5-ht7 receptor, our results indicate that the 5-HT receptor mediating external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs is operationally similar to the putative 5-HT7 receptor mediating relaxation of vascular and non-vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea-pig ileum) as well as tachycardia in the cat.

A common site of action for two allosteric antagonists

A common site of action for two allosteric antagonists

Epipregnanolone acts as a partial agonist on a common neurosteroid modulatory site of the GABA(A) receptor complex in avian CNS.

Neurosteroid modulatory sites present in the GABA(A) receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [3H]flunitrazepam binding with EC50 of 1.18 +/- 0.12 and 6.56 +/- 0.86 microM and Emax of 82.18 +/- 5.80 and 62.98 +/- 3.73%, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC50 of 0.49 +/- 0.15 microM and Emax 12.34 +/- 1.03%. Moreover, the addition of 16 microM epipregnanolone to either allopregnanolone or alphaxalone decreased EC50 values to 0.54 +/- 0.09 and 1.24 +/- 0.25 microM respectively, while Emax values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 microM) of alphaxalone in the presence of allopregnanolone failed to enhance [3H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospecific with regard to the neurosteroid spatial configuration.

Analysis of the bronchoconstrictor responses to adenosine receptor agonists in sensitized guinea-pig lungs and trachea

Airway perfused lungs and half-lungs and superfused tracheal spirals from ovalbumin-sensitized guinea pigs were set up. Adenosine and the analogues, 5′-( N-ethylcarboxamido)adenosine (NECA), R-N 6-phenylisopropyladenosine ( R-PIA), 2-chloroadenosine, N 6-2-(4-aminophenyl)ethyladenosine (APNEA) and 5′-AMP yielded bronchoconstrictor responses as increases in perfusion pressure or of tension, respectively, of these two preparations. These responses were greater in tissues from sensitized compared with un-sensitized guinea pigs. Cross-tachyphylaxis occurred between the constrictor responses to adenosine and the other constrictor adenosine agonists which indicated a common site of action. The adenosine transport inhibitors, dipyridamole and S-(p-nitrobenzyl)-6-thioinosine (NBTI), inhibited the constrictor responses to adenosine and the analogues, except 2-chloroadenosine. This was attributed to a potentiation of the opposing relaxant effects which generally occurred at higher concentrations of the agonists. The P 1 purinoceptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline (A 1 receptor selective) failed to remove the constrictor responses to adenosine either alone or in the presence of dipyridamole. This suggests that the bronchoconstrictor response of sensitized airways tissues is mediated via the novel xanthine-resistant A 3 receptor.

Metal Selectivity of Exocytosis in α‐Toxin‐Permeabilized Bovine Chromaffin Cells

Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal alpha-toxin-permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine beta-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2(+)-evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr(2+)-evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+, Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba(2+)-dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.

Desensitization of the μ‐opioid activation of phospholipase C in SH‐SY5Y cells: the role of protein kinases C and A and Ca2+‐activated K+ currents

1. In SH-SY5Y cells, mu-opioids cause a rapidly desensitizing activation of phospholipase C (PLC), that appears secondary to Ca2+ influx via L-type voltage-sensitive Ca2+ channels (VSCCs). The aim of the present study was to characterize the mechanisms of desensitization of the mu-opioid-induced inositol (1,4,5) triphosphate (Ins(1,4,5)P3) response, by use of a stereospecific radioreceptor mass assay. 2. (R+)-Bay K 8644 (1 nM-10 microM) dose-dependently inhibited fentanyl-induced Ins(1,4,5)P3 formation, with an IC50 of 28.5 nM, confirming our earlier observations that mu-opioids open L-type VSCCs, thus allowing Ca2+ influx to activate PLC. 3. Ro 31-8220 (0.1 nM-10 microM), a protein kinase C inhibitor, dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 20.0 nM), whilst acute phorbol 12,13-dibutrate (1 microM) abolished the response. 4. H-89 (1 nM-10 microM), a protein kinase A inhibitor, also dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 93 nM), whilst dibutryl cyclic AMP (0.5 mM) abolished the response. 5. Blockade of Ca(2+)-activated K+ currents with 4-aminopyridine (2 mM) or iberiotoxin (10 nM) had no effect on fentanyl-induced Ins(1,4,5)P3 formation but further increased the Ro 31-8220-enhanced response. 6. All three mechanisms had additive, or even supra-additive, effects, but only at later (120-300 s) time points. In addition, fentanyl-induced Ins(1,4,5)P3 formation, even if enhanced by H-89, Ro 31-8220 and/or 4-aminopyridine, was inhibited by nifedipine (1 nM-10 microM). 7. In conclusion, desensitization of the mu-opioid-induced activation of PLC is multifactorial, involving protein kinases C and A and Ca(2+)-activated K+ efflux, but the L-type VSCC is of critical importance and may be a possible common site of action.

The 5-HT1-like receptor mediating the increase in canine external carotid blood flow: close resemblance to the 5-HT1D subtype.

1. It has recently been shown that the increase in external carotid blood flow induced by 5-hydroxy-tryptamine (5-HT) in the anaesthetized dog, being mimicked by 5-carboxamidotryptamine (5-CT), inhibited by methiothepin, vagosympathectomy and sympatho-inhibitory drugs, and resistant to blockade by ritanserin and MDL 72222, is mediated by stimulation of prejunctional 5-HT1-like receptors leading to an inhibitory action on carotid sympathetic nerves; these 5-HT1-like receptors are unrelated to either the 5-HT1A, 5-HT1B or 5-HT1C (now 5-HT2C) receptor subtypes. Inasmuch as 5-CT, 5-methoxytryptamine, sumatriptan and metergoline display high affinity, amongst other 5-HT binding sites, for the 5-HT1D subtype, in the present study we have used these drugs in an attempt to determine whether the above inhibitory prejunctional 5-HT1-like receptors correlate with the 5-HT1D subtype. 2. One-minute intracarotid (i.c.) infusions of 5-HT (0.3, 1, 3 and 10 micrograms), 5-CT (0.01, 0.03, 0.1 and 0.3 micrograms), 5-methoxytryptamine (1, 3, 10 and 30 micrograms) and sumatriptan (1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent increases in external carotid blood flow (without changes in mean arterial blood pressure or heart rate) with the following rank order of agonist potency: 5-CT >> 5-HT > 5-methoxytryptamine > or = sumatriptan. Interestingly, sumatriptan-induced vasodilatation was followed by a more pronounced vasoconstriction. 3. The external carotid vasodilator effects of 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan were dose-dependently and specifically antagonized by metergoline (10, 30 and/or 100 micrograms kg-1, i.v.). In addition, 5-methoxytryptamine- and sumatriptan-induced vasodilator effects were, respectively, markedly inhibited or abolished after vagosympathectomy, as previously shown for 5-CT and 5-HT.4. Sumatriptan showed tachyphylaxis in its vasodilator component and antagonized 5-HT-induced external carotid vasodilatation in a specific manner, suggesting that a common site of action may be involved.5. Taken together, the above results support our contention that 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan produce external carotid vasodilatation in the dog by an action that might primarily involve a prejunctional inhibition on carotid sympathetic nerves; a secondary component of this vasodilator response may be postsynaptic (endothelium-dependent and/or even directly on the vasculature).Based on the rank order of agonist potency, inhibition by vagosympathectomy and blockade by metergoline, we suggest that the inhibitory prejunctional 5-HT1-like receptors mediating external carotid vasodilatation in the dog closely resemble the 5-HTID receptor subtype. The pharmacological profile of these receptors is similar (sympathetic nerves of the rat kidney and human saphenous vein, as well as porcine coronary endothelium) to other putative 5-HTID receptors mediating vascular responses.