Modulation of 5-hydroxytryptamine release by presynaptic inhibitory α2-adrenoceptors in the human cerebral cortex

Summary Slices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [³H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of -adrenoceptor agaonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied.In human cerebral cortical slices, the electrically-evoked [³H] overflow was inhibited by noradrenaline (pIC₂₅ value: 6.35); the non-selective -adrenoceptor antagonist phentolamine, at a concentration of 0.32 mol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA₂ value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K⁺-evoked[³H] overflow in a concentration dependent manner; the ₂-adrenoceptor clonidine (1 mol/l), but not the ₁-adrenoceptor agonist methoxamine (1 mol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mol/l) was abolished by the ₂-but not by the ₁-adrenoceptor antagonist prazosin (1 mol/l).It is concluded that release-inhibiting adrenoceptors of the ₂-subtype exist on 5-hydrpxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.Send offprint requests to M. Raiteri at the above address     

Effects of 5-HT released from platelets on thrombin-induced aggregation and ATP release in rabbit pla

Ｅｆｅｃｔｓｏｆ５ＨＴｒｅｌｅａｓｅｄｆｒｏｍｐｌａｔｅｌｅｔｓｏｎｔｈｒｏｍｂｉｎｉｎｄｕｃｅｄａｇｇｒｅｇａｔｉｏｎａｎｄＡＴＰｒｅｌｅａｓｅｉｎｒａｂｂｉｔｐｌａｔｅｌｅｔｓｉｎｖｉｔｒｏＬＩＢａｉＹａｎ１，ＬＩＷｅｎＨａｎ（Ｄｅｐａｒ...     

Dual effects of 5-hydroxytryptamine on stable analogue of thromboxane A~(2~_) induced aggregation and release reaction in rabbit platelets

目的：研究５ＨＴ对ＳＴＡ２血小板聚集和释放反应的影响及可能的分子机制．方法：以透光法，介质中ＡＴＰ含量及荧光图像法评价血小板变形，聚集反应和［Ｃａ２］ｉ水平．结果：（１）５ＨＴ预处理可消除ＳＴＡ２的血小板变形，ＳＴＡ２０３μｍｏｌ·Ｌ－１的聚集增强，ｌ－３μｍｏｌ·Ｌ－１的聚集不变，释放反应抑制．（２）５ＨＴ预处理增加ＳＴＡ２０３μｍｏｌ·Ｌ－１的［Ｃａ２＋］ｉ，降低３μｍｏｌ·Ｌ－１的［Ｃａ２＋］ｉ降低．（３）延长加入５ＨＴ和ＳＴＡ２的间隔，ＳＴＡ２０３μｍｏｌ·Ｌ－１的聚集增强，３μｍｏｌ·Ｌ－１的聚集不变，释放反应抑制．结论：５ＨＴ对ＳＴＡ２介导的聚集和释放反应有双重影响．对ＳＴＡ２［Ｃａ２＋］ｉ的调节可能是上述反应的分子机制．     

α2-Adrenoceptor modulation of rat ventral hippocampal 5-hydroxytryptamine release in vivo

Summary The putative existence of a functional ₂-adrenoceptor-mediated modulation of 5-HT release in vivo from serotonergic neuronal terminals in the ventral hippocampus was investigated using intracerebral microdialysis in chloral hydrate-anaesthetised rats. The ₂-adrenoceptor agonist clonidine (0.01–0.3 mg/kg, SC) dose-dependently decreased the 5-HT output. The response to clonidine was antagonized by systemic or local administration of the ₂-adrenoceptor antagonist idazoxan (0.1 mg/kg, SC, or 10 µmol/l, via the dialysis perfusion medium). Similarly, the 5-HT release-suppressing response to the thiazole ₂-adrenoceptor agonist jingsongling (0.1 mg/kg, SC) was blocked by idazoxan (0.1 mg/kg, SC). The mixed -adrenoceptor/5-HT₁ receptor antagonist pindolol (8.0 mg/kg, SC) did not affect the clonidine-induced reduction of 5-HT release. Tyrosine hydroxylase inhibition by means of -methyl-para-tyrosine (-MT; 250 mg/kg, IP) caused a drastic reduction (>80%) in dialysate 3,4-dihydroxyphenyl acetic acid (DOPAC) output but did not affect the 5-HT output per se over 3 h post-injection. Nor did the -MT pretreatment prevent, but instead significantly enhanced, the 5-HT release-suppressing effect of clonidine. The results demonstrate that the release of 5-HT from serotonergic nerve terminals in rat ventral hippocampus in vivo is modulated by ₂-adrenoceptors, probably both by heteroreceptors on the axon terminals of the serotonergic neurones and by other ₂-adrenoceptor sites situated pre- and/or postsynaptic to the noradrenergic terminals. Our results also suggest that while functionally operative, these sites may receive little physiological tone, at least in chloral hydrate-anaesthetised rats. Send offprint requests to S. Hjorth at the above address     

α2-Adrenoceptor mediated inhibition of the release of radiolabelled 5-hydroxytryptamine and noradrenaline from slices of the dorsal region of the rat brain

Summary Possible local interactions between noradrenergic and serotonergic systems in the dorsal raphe region of the rat were investigated by studying the effects of various drugs on depolarization (20 mmol/l K⁺)-induced release of [³H]5-hydroxytryptamine (5-HT) and [³H]noradrenaline (NA) in vitro using a superfusion method. Exogenous 5-HT did not influence the release of [³H]NA. However, NA (in the presence of 10 mol/l desipramine) as well as the selective ₂-adrenoceptor agonists clonidine and oxymetazoline strongly inhibited [³H]5-HT release. The selective ₁-adrenoceptor agonists phenylephrine and methoxamine did not affect the release of either [³H]5-HT or [⁵H]NA. The inhibition by NA of both [³H]5-HT and [⁵H]NA release was not affected by the -adrenoceptor antagonist sotalol nor by the selective ₁-adrenoceptor antagonist prazosin. However, phentolamine and the selective ₂-adrenoceptor antagonists yohimbine and rauwolscine competitively antagonized the inhibitory effect of NA on [³H]NA release (respective pA₂-values 7.5 and 8.3) and on [³H]5-HT release (respective pA₂-values 7.7 and 8.2). Moreover, the release of [³H]NA and also, but to a lesser extent, that of [³H]5-HT were increased by the antagonists. It is concluded that the release of both 5-HT and NA in the dorsal raphe region may be subject to presynaptic inhibition by NA via activation of ₂-adrenoceptors.Send offprint requests to A. L. Frankhuijzen     

Effect of dopamine on adenosine 3′,5′-cyclic monophosphate levels in human platelets

1. The dopamine effect on intraplatelet adenosine 3′,5′-cyclic monophosphate (cAMP) levels was evaluated in healthy subjects.2. Dopamine levels over 30 nmol/l increased cAMP concentration in a concentration-dependent way.3. The platelet preincubation with propranolol (β-adrenoceptor antagonist) prevented the dopamine effect, whereas phentolamine (α-adrenoceptor antagonist) failed to modify the platelet response to this catecholamine. Dopamine receptor antagonist domperidone directly increased cAMP levels and it enhanced the dopamine effects.4. Our results indicate that dopamine effect is mainly mediated by β-adrenoceptor stimulation.     

Effects of several 5′-carboxamide derivatives of adenosine on adenosine receptors of human platelets and rat fat cells

Summary The effects of several 5-carboxamide derivatives of adenosine on stimulatory (R _a) adenosine receptors of human platelets and inhibitory (R _i) adenosine receptors of rat fat cells have been compared. 5-N-Cyclopropylcarboxamidoadenosine (CPCA) and 5-N-ethylcarboxamidoadenosine (NECA) most potently inhibited ADP-induced aggregation of human platelets as shown by IC₅₀-values of 0.24 and 0.34 mol/l. 5-N-Methylcarboxamidoadenosine (MECA; IC₅₀ 0.81 mol/l) and 5-N-carboxamidoadenosine (NCA; IC₅₀ 2.1 mol/l) were less potent, whereas adenosine, 2-chloroadenosine and (-)N⁶-phenylisopropyladenosine [(-)PIA] exhibit IC₅₀-values of about 1.5 mol/l. Nearly the same rank order of potency was obtained for stimulation of adenylate cyclase activity of platelet membranes and for inhibition of [³H]NECA binding to human platelets. In order to examine the effects of the carboxamide analogues on R _i adenosine receptors of rat fat cells inhibition of lipolysis and adenylate cyclase were studied. (-)PIA was the most potent inhibitor of lipolysis as shown by an IC₅₀ of 0.5 nmol/l, followed by CPCA (IC₅₀ 1.1 nmol/l) and NECA (IC₅₀ 1.3 nmol/l), whereas MECA (IC₅₀ 17.9 nmol/l) and NCA (IC₅₀ 20.1 nmol/l) were much less potent than NECA in inhibiting lipolysis. Similar results were obtained for inhibition of adenylate cyclase activity of fat cell membranes and for competition with [³H]PIA binding to fat cell membranes. The relative potencies of the adenosine analogues at both receptor subclasses were calculated from the ratio of the IC₅₀-values for inhibition of platelet aggregation and of lipolysis. (-)PIA showed the highest selectivity for R _i receptors as indicated by a 2,900-fold lower IC₅₀ for the antilipolytic than for the antiaggregatory effect. The R _a/R _i activity ratio for NECA was about 260, for CPCA 220, for NCA 105 and for MECA 45. These results indicate that all 5-carboxamide adenosine derivatives are more potent agonists at R _i receptors than at R _a receptors. Since MECA has a higher selectivity for R _a receptors than NECA, it may be useful for the characterization of stimulatory adenosine receptors in different tissues.     

β-Endorphin-induced locomotor stimulation and reinforcement are associated with an increase in dopamine release in the nucleus accumbens

In vivo microdialysis was used to compare the effects of β-endorphin upon dopamine (DA) release in the nucleus accumbens (NAC) of anesthetized versus freely moving rats, and to examine the role of the the mesolimbic DA system in mediating both the motoric and secondary reinforcing effects of this peptide. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), using a reversed phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (ICV) administration of β-endorphin (2.5 and 5.0 µg) increased DA release and metabolites in both freely moving and anesthetized rats. This effect was of greater magnitude and duration in freely moving rats and was accompanied by stimulation of locomotor activity. The 5 µg dose also functioned as a secondary reinforcer in a conditioned place preference paradigm. A higher dose of β-endorphin (7.5 µg) stimulated DA release and metabolites in anesthetized rats but failed to affect these parameters in freely moving rats. At this dose, catalepsy and a loss of the reinforcing effects of this peptide were observed. These data demonstrate marked differences in the effects of β-endorphin upon DA release in the awake versus anesthetized rat. Further, the finding that the reinforcing and locomotor stimulating effects of β-endorphin only occur at those doses which stimulate DA release suggest that this action is critical for the expression of both behavioral effects.     

Differential effects of electrical stimulation,blockade of neuronal amine uptake and activation of α2-adrenoceptors on the release of endogenous noradrenaline and 5-hydroxytryptamine from the isolated rat pineal gland

Summary Isolated rat pineal glands were incubated in vitro and the release of endogenous noradrenaline or 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection. In the absence of test drugs, the spontaneous outflow of noradrenaline was about 10 fmol/10 min and electrical stimulation (5 Hz, 1500 pulses) evoked the release of about 70 fmol noradrenaline. Nomifensine enhanced the spontaneous outflow of noradrenaline about threefold and the electrically evoked release of noradrenaline about sixfold. In the presence of nomifensine, the ₂-adrenoceptor antagonist yohimbine markedly increased the electrically evoked release of noradrenaline, whereas the ₁-adrenoceptor antagonist prazosin had no effect. Clonidine inhibited the electrically evoked release of noradrenaline by about 65%, and this was antagonized by yohimbine in a competitive manner. In the absence of drugs, the initial spontaneous outflow of 5-HT was (compared with noradrenaline) very high 64 mol/10 min. It declined by 80% within 1 h of incubation in vitro. The outflow of 5-HIAA amounted initially to 38 mol/10 min and declined by 40% within 1 h of incubation. Addition of l-tryptophan (10 mol/1) after 1 h of incubation in vitro largely enhanced the outflow of 5-HT and 5-HIAA within 30 min of incubation (about ten- and fourfold, respectively). When l-tryptophan was present from the onset of incubation the initial outflow of 5-HT and 5-HIAA was only slightly elevated, but the decline was largely attenuated. Neither omission of calcium nor addition of nomifensine, clonidine or yohimbine significantly affected the spontaneous outflow of 5-HT or 5-HIAA. Likewise, neither electrical stimulation in the absence or presence of nomifensine and yohimbine nor stimulation by high potassium (45 mmol/1) significantly affected the outflow of 5-HT or 5-HIAA.In conclusion, the release of endogenous noradrenaline from the sympathetic nerves terminating in the pin eal gland is inhibited by presynaptic ₂-adrenoceptors. The outflow of 5-HT from the pineal gland originates almost exclusively from non-neuronal cells, most probably the pinealocytes, and depends largely on a continuous de novo synthesis. Catabolism of 5-HT to 5-HIAA in the pineal gland occurs mainly in an extraneuronal compartment, probably the pinealocytes and/or the interstitial cells of the pineal gland. Send offprint requests to K. Racké at the above address     

Roflumilast inhibits the release of chemokines and TNF-α from human lung macrophages stimulated with lipopolysaccharide

BACKGROUND AND PURPOSE

Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N-oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF-α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

EXPERIMENTAL APPROACH

Lung macrophages isolated from resected human lungs were incubated with roflumilast, roflumilast N-oxide, PGE₂, the COX inhibitor indomethacin, the COX-2 inhibitor NS-398 or vehicle and stimulated with LPS (24 h). Chemokines, TNF-α, PGE₂ and 6-keto PGF_1α were measured in culture supernatants by immunoassay. COX-2 mRNA expression was assessed with RT-qPCR. PDE activities were determined in macrophage homogenates.

KEY RESULTS

Expression of PDE4 in lung macrophages was increased after incubation with LPS. Roflumilast and roflumilast N-oxide concentration-dependently reduced the LPS-stimulated release of CCL2, CCL3, CCL4, CXCL10 and TNF-α from human lung macrophages, whereas that of CXCL1 or CXCL8 was not altered. This reduction by the PDE4 inhibitors was further accentuated by exogenous PGE₂ (10 nM) but abolished in the presence of indomethacin or NS-398. Conversely, addition of PGE₂ (10 nM), in the presence of indomethacin restored inhibition by roflumilast. LPS also increased PGE₂ and 6-keto PGF_1α release from lung macrophages which was associated with an up-regulation of COX-2 mRNA.

CONCLUSIONS AND IMPLICATIONS

Roflumilast and roflumilast N-oxide reduced LPS-induced release of CCL2, 3, 4, CXCL10 and TNF-α in human lung macrophages.     

β-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5′-triphosphate release from sympathetic nerves supplying the rat tail artery

1. The effects of prejunctional β-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5′-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro.
2. Isoprenaline (0.1 μM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train.
3. Isoprenaline (0.1 μM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps.
4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the β-adrenoceptor antagonist, propranolol (0.1 μM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents.
5. Thus, activation of prejunctional β-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.

     

Agonistic and antagonistic effects of various α-adrenergic agonists in human platelets

Summary In human platelets, the effects of various -adrenergic agonists were studied on platelet aggregation and adenylate cyclase activity. Out of many phenylethylamine derivatives tested, only some catecholamines were able to act as -adrenergic agonists inducing platelet aggregation and inhibition of adenylate cyclase with the order of potency: adrenaline > noradrenaline>-methylnoradrenaline. Other phenylethylamine and imidazoline derivatives, which act as potent -adrenergic agonists in various systems, neither induced primary aggregation nor adenylate cyclase inhibition, when tested at concentrations up to 1 mM. Since binding studies indicated high affinities of these agents to the platelet -adrenergic receptor, their effects on adrenaline-induced aggregation and adenylate cyclase inhibition were studied.Both types of -adrenergic agonists tested, phenylethylamine and imidazoline derivatives, prevented adrenaline-induced aggregation and adenylate cyclase inhibition. The imidazolines, xylometazoline, oxymetazoline, naphazoline, clonidine and tetryzolin, were the most effective antagonists with similar potencies as observed with the typical -adrenergic antagonists, phentolamine and yohimbine. Phenylethylamine derivatives such as phenylephrine, methoxamine, synephrine and norfenefrine, similarly antagonized the adrenaline-induced responses but higher concentrations were required. The potencies of these phenylethylamine derivatives were similar to those of the classical -adrenergic antagonists, phenoxybenzamine and azapetine. The results indicate that the platelet -adrenergic receptor, which has many similarities with the ₂-adrenergic receptor with regard to affinities of various -adrenergic agonists, completely differs from that found in other tissues, inasmuch as only some catecholamines acted as agonists whereas other phenylethylamine derivatives and imidazoline derivatives acted as antagonists.A preliminary report of these studies has been presented (Jakobs, 1978)     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address     

Enhancement of ADP-induced aggregation by 5-HT in rabbit platelets

目的：研究５ＨＴ对ＡＤＰ介导的血小板聚集反应的增强作用．方法：以透光法、图像法和受体结合法评价聚集反应、单细胞内钙和三磷酸肌醇的含量．结果：５ＨＴ（００３－３μｍｏｌ·Ｌ－１）浓度依赖性地引起ＰＲＰ的透光度降低（ＤＬＴ），电镜结果显示血小板变形的同时伴有颗粒中心化，无聚集和释放反应．Ｆｕｒａ２负载后，５ＨＴ升高［Ｃａ２＋］ｉ，９０秒达峰值，ＩＰ３一过性升高．ＡＤＰ同样引起ＤＬＴ，但可被５ＨＴ消除，呈浓度依赖性．ＡＤＰ的聚集反应和［Ｃａ２＋］ｉ动员则由于５ＨＴ预处理而升高．结论：５ＨＴ增强ＡＤＰ的聚集反应与５ＨＴ的细胞内钙动员及ＡＤＰ的外钙内流两者的叠加作用有关．     

Interleukin 8 modulates interleukin-1β, interleukin-6 and tumor necrosis factor-α release from normal human mononuclear cells

Recombinant human interleukin 8 (IL-8) enhanced the release of inflammatory cytokines including interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) from normal human mononuclear cells in a dose-related manner (from 1 ng/ml to 10 ng/ml with a maximal effect at 5 ng/ml( when the cells incubated with IL-8 for 24 h. This cytokine-releasing activity of IL-8 is temperature-dependent and required protein synthesis since low temperature (4°C) and cycloheximide (100 μg/ml) minimized the cytokine release from MNC. However, when IL-8 concentration was greater than 20 ng/ml, the cytokine release was supressed. For further investigating the subcellular mechanism of the adverse effect of high dose IL-8 (20 ng/ml) in cytokine synthesis, human mononuclear cells (1 × 10⁶/ml) were stimulated with PHA (1 μg/ml) in the presence of 20 ng/ml IL-8 for 3 days. We found not only [³H]thymidine incorporation of MNC was tremendously inhibited but DNA fragmentation appeared. Subsequently, the cell cycle of PHA-stimulated MNC retarded in the phase of G₀/G₁. These results suggest that in low concentration (5–10 ng/ml) IL-8 not only activated neutrophil phagocytosis but facilitated the release of inflammatory cytokines from mononuclear cells. Higher dose of IL-8 (more than 20 ng/ml) conversely suppressed these cytokine release from damaged cells by its cytotoxic effect. This newly found cytokine-releasing activity of IL-8 may play a role in the modulation of inflammation.     

Kinetics of drug release from polylactic acid—hydrocortisone microcapsules

Abstract

Polylactic acid microcapsules of similar particle size distribution containing various drug loadings of hydrocortisone were prepared. The microcapsules, which contained randomly dispensed drug particles, showed a dissolution pattern which consists of a fast first-stage and a slow second-stage drug release. Our studies showed that the kinetics of drug release from the microcapsules can be adequately described by a spherical matrix model based on a flux mechanism involving the diffusion of dissolved drug at the penetrating front of the dissolution medium. Drug loading played an important role in the control of drug release rate. An empirical relationship between drug loading and drug diffusi-bility through the polymeric matrix was developed and showed that the rate of drug release increased exponentially with the increase in drug loading. The microcapsules were further shown to exhibit increased rate of drug release in dissolution medium containing either cetylpyridium chloride or aerosol OT. The effect of the surfactants was attributed to surface tension lowering and improved wetting of the microcapsule particles.     

Superoxide radicals increase transforming growth factor-β1 and collagen release from human lung fibroblasts via cellular influx through chloride channels

Reactive oxygen species (ROS) have been implicated in the pathogenesis of fibrosis. However, it remains unclear which ROS is the major cause. We hypothesize that superoxide elicits specific toxicity to human lung fibroblasts and plays an important role in the development of pulmonary fibrosis. In this study, superoxide generated from xanthine and xanthine oxidase activated lung fibroblasts by increasing the release of TGF-β1 and collagen. This was associated with increased levels of intracellular superoxide. SOD and tempol, by scavenging respectively extracellular and intracellular superoxide, prevented the activation of fibroblasts induced by exposure to exogenous superoxide, whereas catalase did not. Moreover, hydrogen peroxide did not activate fibroblasts. Apparently, superoxide rather than hydrogen peroxide is involved in the regulation of TGF-β1 and collagen release in lung fibroblasts. The chloride channel blocker, DIDS, inhibited the increase of intracellular superoxide levels induced by exogenous superoxide and consequently prevented the activation of fibroblasts. This suggests that the cellular influx of superoxide through chloride channels is essential for superoxide-induced activation of fibroblasts. ERK1/2 and p38 MAPKs are involved in the intracellular pathway leading to superoxide-induced fibroblasts activation.Superoxide possesses until now undiscovered specific pro-fibrotic properties in human lung fibroblasts. This takes place via the cellular influx of superoxide through chloride channels rather than via the formation of hydrogen peroxide.     

Comparison of ibuprofen release from minitablets and capsules containing ibuprofen: β-Cyclodextrin complex

Mixtures containing ibuprofen (IB) complexed with β-cyclodextrin (βCD) obtained by two complexation methods [suspension/solution (with water removed by air stream, spray- and freeze-drying) and kneading technique] were processed into pharmaceutical dosage forms (minitablets and capsules). Powders (IB, βCD and IBβCD) were characterized for moisture content, densities (true and bulk), angle of repose and Carr’s index, X-ray and NMR. From physical mixtures and IBβCD complexes without other excipients were prepared 2.5-mm-diameter minitablets and capsules. Minitablets were characterized for the energy of compaction, tensile strength, friability, density and IB release (at pH 1.0 and 7.2), whereby capsules were characterized for IB release. The results from the release of IB were analyzed using different parameters, namely, the similarity factor (f₂), the dissolution efficiency (DE) and the amounts released at a certain time (30, 60 and 180 min) and compared statistically (α = 0.05). The release of IB from the minitablets showed no dependency on the amount of water used in the formation of the complexes. Differences were due to the compaction force used or the presence of a shell for the capsules. The differences observed were mostly due to the characteristics of the particles (dependent on the method considered on the formation of the complexes) and neither to the dosage form nor to the complex of the IB.     

Presynaptic α2-adrenoceptors mediating inhibition of noradrenalh and 5-hydroxytryptamine release in rat cerebral cortex: further characterization as different α2-adrenoceptor subtynes

Summary In a previous investigation it was suggested that the ₂-adrenoceptors regulating ³H-noradrenaline (³H-NA) release and the ₂-heteroreceptors regulating the release of ³H-5-hydroxytryptamine (³H-5-HT) from rat cerebrocortex synaptosomes represent different subtypes of the ₂-adrenoceptor in that (–)-mianserin potently blocked the receptors sited on 5-HT terminals but was ineffective at the autoreceptors (Raiteri et al. 1983).In this work a number of ₂-adrenoceptor antagonists were tested against NA as an inhibitor of the K⁺ (15 mmol/l)-evoked release of ³H-NA or ³H-5-HT (in presence of 1 mol/l desipramine or citalopram, respectively) from superfused rat neocortex synaptosomes. The order of apparent affinity of the antagonists was: idazoxan ORG 20769 (2-amino-4-(1-methyl-1,2,3, 6-tetrahydropyridin-4-yl)-thiazole-5-carbonitrile (Z)-2-butenedioate (1:1) salt) ORG 20350 (5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (Z)-2-butenedioate (1:1) salt) ORG 20091 (5-chloro-4-(1-methyl -1,2,5,6-tetrahydropyridin -3- yl)-thiazole-2-amine (Z)-2-butenedioate (2:1) salt) at the ₂-autoreceptor and idazoxan ORG 20769 > ORG 20091 » ORG 20350 at the ₂-heteroreceptor. Prazosin (1 gmol/l) or AR-C 239 (1 gmol/l) (2-[2-[4-(o-methoxyphenyl)piperazine-l-yl]ethyl]-4,4-dimethyl-1,3(2H,4H)isoquinolinedione) were ineffective in both systems.Idazoxan and ORG 20769 had a comparable apparent affinity at the ₂-autoreceptor (pK_B values: 8.45 and 8.42, respectively) and at the heteroreceptor (pK_B values: 8.16 and 8.15, respectively). In contrast, ORG 20350 was about 14-fold less. potent than the two previous compounds at the autoreceptor (pKB = 7.30) whereas it was ineffective at the heteroreceptor when tested up to 3 mol/l (pK_B < 5.5). Experiments with electrically-stimulated (2 Hz; 2 ms; 24 mA) rat cerebral cortex slices confirmed the data with synaptosomes. ORG 20350 shifted to the right in a parallel manner the concentration-response curve of clonidine at the ₂-autoreceptors (pA₂ = 7.25). The sulphydryl alkylating agent N-ethyl-maleimide (NEM; 3 mol/l) which has been proposed to inactivate pertussis toxin sensitive G proteins, abolished the inhibition of both ³H-NA and ³H-5-HT release caused by the ₂-adrenoceptor agonist clonidine (0.3 mol/l) in hippocampus synaptosomes. The effect of exposure to NEM was not modified during protection experiments with idazoxan.The results lend further support to the proposed existence of functional ₂-adrenoceptor subtypes. It should be noted that the two pharmacologically distinct receptors here characterized are present in the same brain area and within the same animal species. They are sited on the axon terminals of different neurons. Their function appears that of inhibiting NA or 5-HT release, respectively. Both ₂-auto- and heteroreceptors are likely to be coupled to G proteins. According to the current nomenclature, the receptors do not seem to belong to the _2B subtype. However only one of them might be classified as _2A. Send offprint requests to M. Raiteri at the above address     

Effects of MDL 72222 and methiothepin on carotid vascular responses to 5-hydroxytryptamine in the pig: Evidence for the presence of “5-hydroxytryptamine1-like” receptors

Summary The present study concerns the effects of MDL 72222 (0.5 mg · kg^–1, i.v.), a 5-hydroxytryptamine₃ (5-HT₃) receptor antagonist, and methiothepin (1.0 mg · kg^–1, i.v.), an antagonist of both 5-HT₂ and 5-HT₁-like receptors, on the responses to local infusions of 5-HT (2.0 g · kg^–1 · min^–1) on the total common carotid artery blood flow and its complete distribution in anaesthetized pigs. As reported earlier, more than 80% of the carotid blood bypassed the capillary circulation via cranial arteriovenous anastomoses, while approximately 15% and 2% was distributed to the extracerebral structures and brain, respectively. The total carotid blood flow did not change or was moderately reduced by 5-HT, but the amine consistently caused a 85% reduction in arteriovenous anastomotic blood flow and a 5-fold increase in blood flow to the extracerebral tissues, mainly the skin and ears. The colour of the skin and ears changed to bright pink. Complete recovery from the effects of 5-HT was observed once the infusion was stopped. MDL 72222 and methiothepin did not themselves affect carotid haemodynamics. The responses to 5-HT were not modified by MDL 72222 except that the reduction of the total carotid blood flow by 5-HT was augmented. In contrast, methiothepin almost completely abolished both the reduction of arteriovenous anastomotic blood flow and the increase in tissue blood flow following 5-HT-infusion. The colour of the skin and ears also did not become pink. In conjunction with our earlier findings that the constriction of arteriovenous anastomoses and the dilatation of arterioles caused by 5-HT within the carotid territory of the pig are not attenuated by 5-HT₂ receptor antagonists (cyproheptadine, methysergide, ketanserin and WAL 1307), and are mimicked by agonists at 5-HT₁-like receptors (5-carboxamidotryptamine and BEA 1654), our results clearly establish that these responses are mediated by 5-HT₁-like receptors.