查看更多>>摘要：Deorphanised G-protein-coupled receptors represent new and expanding targets for drug development. Neuropeptide B (NPB) and W (NPW) have recently been identified as the cognate endogenous ligands for the orphan receptor GPR7, now designated as NPBW_1. NPB and NPW also bound to a second related orphan receptor, GPR8, now designated as NPBW_2 that is present in humans but not rats or mice. In humans, high levels of NPW mRNA have been visualised in the substantia nigra, whereas moderate expression levels have been detected in the amygdala and hippocampus. In peripheral tissues, expression of NPW mRNA has been confirmed in the progenital system, comprising the kidney, testis, uterus, ovary and placenta, and also in stomach homogenates. Immunocytochemical, molecular biological and autoradiography techniques have revealed a discrete CNS distribution for NPBW_1 in human, mouse and rat. Highest expression of NPBW_1 mRNA and protein was identified in the amygdala and hypothalamic nuclei known to regulate feeding behaviour. [~(125)I]-NPW bound with a single high affinity to rat amygdala, K_D = 0.44 nM and 150 fmol mg~(-1) protein. Physiological studies demonstrate that intracerebroventricular infusion of NPBW_1 ligands modulates feeding behaviour, regulates the release of corticosterone, prolactin and growth hormone while also manipulating pain pathway. Mouse knockout models of the gene encoding either NPB or NPBW_1 have a gender-specific phenotype, with moderate obesity evident in males but not females. Further investigation is required to elucidate the precise physiological role of NPB and NPW as neurotransmitters.
Mauro A.M. CaraiGiancarlo ColomboGian Luigi GessaRatnakumar Yalamanchili...
p.1043-1050页
查看更多>>摘要：1 This study investigated whether (a) cannabinoid CB_1 receptor knockout (CB_1~(-/-)) mice displayed altered gastrointestinal transit and (b) cannabinoid CB_1 and opioid receptors functionally interact in the regulation of gastrointestinal transit. 2 Gastrointestinal transit was assessed by the Whole Gastrointestinal Transit, measuring the excretion time of an intragastrically administered marker (whole intestine), and the Upper Gastrointestinal Transit, measuring the distance covered by the marker in the small intestine. 3 CB_1~(-/-) and homozygous CB_1~(+/+) (CB_1~(+/+)) mice did not differ in both whole gut and small intestine transit. CB_1~(-/-) and CB_1~(+/+) mice were equally responsive to the inhibitory effect of morphine (10 mg kg~(-1)) and loperamide (3 mg kg~(-1)) on whole gut transit. 4 Additionally, in CD1 mice the cannabinoid CB_1 receptor antagonist, rimonabant (0-0.5 mg kg~(-1)), failed to block the inhibitory effect of morphine (0-1.25 mg kg~(-1)) and loperamide (0-0.5 mg kg~(-1)) on transit in small and whole intestine. Similarly, the opioid receptor antagonists, naloxone (0-1 mg kg~(-1)) and naltrexone (0-10 mg kg~(-1)), failed to block the inhibitory effect of the cannabinoid WIN 55,212-2 (0-3 mg kg~(-1)) on transit in small and whole intestine. 5 These results suggest that (a) compensatory mechanisms likely developed in CB_1~(-/-) mice to overcome the lack of inhibitory function of endocannabinoid system; (b) cannabinoid and opioid receptor systems did not interact in regulating gastrointestinal transit in mice.
查看更多>>摘要：1 In the light of recent findings that VPAC1 and VPAC2 receptors form homodimers and heterodimers, we have evaluated the function of these receptors coexpressed in the same cells, using whole-cell and membrane preparations. Cells expressing each receptor alone were used for comparison. 2 The study was performed on Chinese hamster ovary cells stably transfected with both human recombinant receptors and we compared receptor occupancy and adenylate cyclase activation by VIP, Ro 25-1553 - a VPAC2 selective agonist - and [K~(15),R~(16),L~(27)]VIP(1-7)/GRF(8-27) - a VPAC1 selective agonist - on membranes prepared from each cell line and on a mixture of membranes from cells expressing each receptor individually. We also studied receptor internalization induced by the three agonists on intact cells expressing both receptors alone or together by fluorescence-activated cell sorting using monoclonal antibodies and demonstrated by using co-immunoprecipitation that the two receptors did interact. 3 The results indicated that coexpression of the receptors did not modify the recognition of ligands, nor the capacity of the agonists to stimulate adenylate cyclase activity and, in intact cells, to induce internalization of the receptors. 4 As a consequence, the properties of the selective ligands that were established on cell lines expressing a single population of VIP receptors were valid on cells expressing both receptors. Furthermore, the recently demonstrated VPAC1/VPAC2 receptor heterodimerization did not affect the function of either receptor.
Susana E. MorenoJose C. Alves-FilhoGiuliana BertoziTais M. Alfaya...
p.1060-1066页
查看更多>>摘要：1 Interleukin-2 (IL-2) has proinflammatory properties that limit its therapeutic use. Its side effects are mainly explained by the induction of a vascular leakage syndrome. Cytokines, as TNF-α and IL-1β, and nitric oxide (NO) generated by IL-2-activated leukocytes play a role in this defect. 2 As the systemic release of these mediators inhibits neutrophil migration to a specific inflammatory site, we investigated now whether IL-2 administrated systemically inhibits the neutrophil recruitment to the inflamed peritoneum. The involvement of NO in the process was also addressed. 3 Using peritoneal neutrophils, we show that the intravenous treatment of the mice with IL-2 inhibits the neutrophil migration induced by carrageenin, LPS or fMLP. In confirmation, IL-2-treated mice showed a significant reduction in leukocyte rolling and adhesion in mesenteric microcirculation evaluated after carrageenin, LPS and fMLP injections. Aminoguanidine prevented the inhibitory effect of IL-2 on carrageenin-induced neutrophil migration, rolling and adhesion. In contrast, IL-2 failed to reduce the lung leukocyte infiltration induced by LPS. Therefore, IL-2 inhibition of neutrophil migration is organ specific. 4 Our results indicate that IL-2 administered systemically inhibits neutrophil recruitment to some inflammatory sites through a mechanism dependent on NO. The results also reinforce the needs to determine the mechanism by which patients treated with IL-2 show increased risks of infection.
Mark J. Paul-ClarkShaun K. Mc MasterElizabeth BelcherRosalinda Sorrentino...
p.1067-1075页
查看更多>>摘要：1 Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFα are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFα induction. 2 In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFa release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam_3CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFα without an effect on NO. 3 At later time points (48-72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFα-binding antibodies. 4 At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. 5 Using cells from TLR2~(-/-) or TLR4~(-/-) mice, the ability of LPS to synergise with S. aureus or Pam_3CSK4 was found to be dependent on both TLR2 and TLR4. 6 These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFα genes compared with their coinduction when both receptor pathways are activated.
Ayten A. GurbanovaRezzan AkerVernal BerkmanFiliz Yilmaz Onat...
p.1076-1082页
查看更多>>摘要：1 Spontaneous 7-10 Hz spike-wave discharges (SWDs) are the electroencephalographic hallmark of absence seizures, as can be observed in WAG/Rij as well as in GAERS, two commonly used well-validated genetic rat models of absence epilepsy. A local upregulation of sodium channels within the perioral region of the primary somatosensory cortex indicated an initiation site for SWDs in WAG/Rij rats, in line with a new theory that assumes that SWDs have a cortical focal origin in the perioral region of the somatosensory cortex. We tested whether bilateral microinfusion at this focal site of the sodium channel blocker phenytoin, which is known to aggravate SWDs after systemic administration, reduces SWDs in both models. 2 WAG/Rij rats and GAERS, chronically provided with cortical EEG electrodes and bilateral cortical cannula's, were used. The EEGs were recorded before and after or systemic or bilateral infusion of phenytoin. 3 Microinfusion of phenytoin at the perioral region of the somatosensory cortex produced an immediate cessation of seizure activity in WAG/Rij rats, while systemic injection produced an increase in both genetic models. Microinfusion of the same and higher concentrations of phenytoin in GAERS at the same stereotactic coordinates showed no effect. Phenytoin was effective in GAERS 2 mm more posteriorly. 4 The data suggest that both genetic models have a cortical area at which diametrically opposite effects of phenytoin can be found compared to systemic injections: a decrease after local microinfusion and aggravation after systemic administration, although the exact cortical location may be different. Moreover, a deficit in sodium channels might be an ethiological factor underlying an increased probability for the initiation of SWDs in the somatosensory cortex.
查看更多>>摘要：1 We previously demonstrated that p-chloroamphetamine (PCA) intravenously (i.v.) evokes a specific patterned bursting response in the vas deferens nerve (VDN) of anaesthetised male rats that is associated with contraction of the vas deferens, and ejaculation and contraction of the bulbospongiosus muscles. The present study used selective 5-HT agonists to induce similar rhythmic bursting responses in the VDN in order to reveal the 5-HT receptor subtypes involved. 2 The 5-HT_(2C) receptor agonist (1.0 mg kg~(-1) Ro600175 i.v.) evoked the characteristic bursting pattern responses in the VDN. The 5-HT_(1A) receptor agonist (1.0 mg kg~(-1) 8-OH-DPAT i.v.) failed to elicit any responses. However, 8-OH-DPAT coadministered in combination with Ro600175 induced a potentiation of the responses. 3 Responses were also evoked in rats with a mid-thoracic spinalisation, with a more predictable response being observed following the combination of agonists. This suggests an action of both agonists in the lumbosacral spinal cord. 4 Responses were blocked by 0.5 mg kg~(-1) SB206553 i.v. (5-HT_(2B/C) receptor antagonist) or 0.5 mg kg~(-1) WAY100635 i.v. (5-HT_(1A) receptor antagonist), but not 0.1 or 1.0 mg kg~(-1) SB269970 i.v. (5-HT_7 receptor antagonist). 5 We suggest that activation of 5-HT_(2C) and 5-HT_(1A) receptor subtypes synergistically elicits contraction of the vas deferens through the activation of sympathetic preganglionic neurones in the spinal cord. 6 These data support the idea of a proejaculatory action of 5-HT_(2C) receptors in the lumbosacral spinal cord, suggesting a descending 5-HT excitatory pathway in addition to a 5-HT inhibitory pathway. An excitatory action of 8-OH-DPAT at lumbosacral sites is also evident.
Charles PearmanWilliam KentNicolas BrackenMunir Hussain...
p.1091-1098页
查看更多>>摘要：1 Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K~+ current (I_(to)) and inward rectifying K~+ current (I_(K1)) in rat cardiac muscle. 2 Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 μmol 1~(-1). A similar degree of inhibition was observed when I_(to) and I_(K1) were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. 3 The IC_(50) was 35.8 μmol 1~(-1) for I_(to) and 27.8 μmol 1~(-1) for I_(K1) compared to 5.4 μmol 1~(-1) for L-type Ca~(2+) current (I_(Ca)). The Hill coefficients for I_(to), I_(K1) and I_(Ca) were -1.97, -1.60 and -1.21, respectively. In addition to inhibiting I_(to) amplitude, H-89 also accelerated the time to peak and the rate of voltage-dependent inactivation so that the time course of I_(to) was abbreviated. 4 Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentration-dependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 μmol 1~(-1)) used. 5 In experiments investigating the effects of elevated cyclic AMP, the β-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on I_(to) or I_(K1). 6 Data suggest that the effects of H-89 on K~+ currents are more complex than simple inhibition of PKA-mediated phosphorylation.
查看更多>>摘要：1 Although there is increasing evidence showing that boswellic acid might be a potential anticancer agent, the mechanisms involved in its action are unclear. 2 In the present study, we showed that acetyl-keto-β-boswellic acid (AKBA) inhibited cellular growth in several colon cancer cell lines. Cell cycle analysis by flow cytometry showed that cells were arrested at the G1 phase after AKBA treatment. 3 Further analysis showed that cyclin D1 and E, CDK 2 and 4 and phosphorylated Rb were decreased in AKBA-treated cells while p21 expression was increased. 4 The growth inhibitory effect of AKBA was dependent on p21 but not p53. HCT-116 p53~(-/-) cells were sensitized to the apoptotic effect of AKBA, suggesting that p21 may have protected cells against apoptosis by inducing a Gl arrest. 5 In conclusion, we have demonstrated that AKBA inhibited cellular growth in colon cancer cells. These findings may have implications to the use of boswellic acids as potential anticancer agents in colon cancer.
Nicolas BrackenMoutaz ElKadriGeorge HartMunir Hussain...
p.1108-1115页
查看更多>>摘要：1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca~(2+) current (I_(Ca)) observed in the absence of exogenous β-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell I_(Ca) in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 μmol 1~(-1)) increased basal I_(Ca) (P < 0.05), whereas H-89 inhibited I_(Ca) in a concentration-dependent manner with an IC_(50) ～5 μmol 1~(-1). H-89 also inhibited the response to 1.0 μmol 1~(-1) isoprenaline, although relatively high concentrations (30 μmol 1~(-1)) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 μmol 1~(-1) H-89 on time-dependent recovery of I_(Ca) from voltage-dependent inactivation as well as the steady-state gating of I_(Ca). T_(0.5) (time for I_(Ca) to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P < 0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of I_(Ca) were unaffected by 10 μmol 1~(-1) H-89 or 1 μmol 1~(-1) calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V_(0.5) for activation and inactivation became more negative. 5 Data show that basal I_(Ca) is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied β-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to β-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.
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