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Biomedical subjects

D Smart

Publications and source records attributed to D Smart.

At least 55 records · Page 3Linked to original sources
PubMed1999

Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2> or =NC=NC(1-13)NH2>NC(1-12)NH2> >NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

Animals↗
PubMed1999

Characterization of recombinant human orexin receptor pharmacology in a Chinese hamster ovary cell-line using FLIPR.

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03+/-0.08 and 7. 30+/-0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18+/-0.10 and 8. 43+/-0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6 - 8 s), followed by a gradually declining secondary phase. Thapsigargin (3 microM) or U73122 (3 microM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx.

Aniline Compounds↗
PubMed1999

The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells.

1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM. 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9. 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively. 4 E-1/E-2 (1 microM) increased [Ca2+]i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adherent cells E-1/E-2 (1 microM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells. 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor.

Analgesics, Opioid↗
PubMed1999

Molecular and pharmacological characterization of a functional tachykinin NK3 receptor cloned from the rabbit iris sphincter muscle.

1. A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. 2. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively. 3. In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKA>/=SP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7 - 0.8), suggesting the possibility of multiple affinity states of the receptor. 4. Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography. 5. Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and 'atypical' NK3 receptor subtypes previously reported.

Amino Acid Sequence↗
PubMed1999

Mental health triage in emergency medicine.

OBJECTIVE: The aim of this study was to: (i) develop a triage scale consistent with the National Triage Scale (NTS) for patients with mental health problems attending emergency departments; and (ii) to reduce emergency waiting times, transit times and improve skills assessing mental health problems. METHOD: We developed a Mental Health Triage Scale (MHTS) consistent with the NTS. The MHTS was then implemented using a structured education package, and evaluated from March to August 1994. Further evaluation occurred after 2 years. RESULTS: A four-tiered MHTS was produced: category 2, violent, aggressive or suicidal, danger to self or others or with police escort; category 3, very distressed or psychotic, likely to deteriorate, situational crisis, danger to self or others; category 4, long-standing semi-urgent mental health disorder, supporting agency present; and category 5, long-standing non-acute mental health disorder, no support agency present. Patients with illness, injury or self-harm were triaged using combined mental health and medical information. Mean emergency waiting times and transit times were reduced. More consistent triaging for mental health patients occurred, and more consistent admission rates by urgency. Reduced mental health 'did not waits' showed improved customer satisfaction. Mental Health Triage Scale was considered appropriate by liaison psychiatry and its use has continued at 2 years follow-up. CONCLUSIONS: A systematic approach to mental health triaging produced a workable scale, reduced waiting times, transit times, and provided effective and consistent integration of mental health patients into a general emergency department.

Australia↗
PubMed1999

Neither nociceptin nor its receptor are present in human synovial fluid or tissue.

Our aim was to identify the nociceptin receptor and its endogenous ligand, nociceptin, in human peripheral tissue. Synovial tissue was obtained from 11 patients (ASA I-III, 66-84 yr) undergoing elective total knee replacement. Synovial fluid was obtained from another 10 patients (ASA I-III, 57-81 yr). Fluid was mixed with trifluoroacetic acid and the tissue with isopentone before freezing at -70 degrees C. Nociceptin receptor identification was performed using a [3H]nociceptin binding assay and nociceptin detection by radioimmunoassay. There was no specific [3H]nociceptin binding to knee synovial tissue and radioimmunoassay did not detect nociceptin. Neither the nociceptin receptor nor nociceptin was found in human synovial tissue or fluid.

Aged↗
PubMed1999

The interleukin-1 type I receptor is expressed in human hypothalamus.

Several lines of evidence suggest that interleukin-1 (IL-1) acts directly on the central nervous system, probably within the hypothalamus, causing effects such as fever, activation of the immune response and sickness behaviour. IL-1 has also been shown to be involved in the aetiology of several neuronal diseases, including neurodegeneration, stroke and Alzheimer's disease. However, the question as to whether the full-length type I IL-1 receptor (IL-1RI) is expressed in the human hypothalamus has yet to be addressed. Using the polymerase chain reaction, we cloned a full-length cDNA encoding the human hypothalamic IL-1RI from human hypothalamic cDNA. The DNA sequence of the human hypothalamic receptor was identical to that of the human fibroblast IL-1RI. The IL-1RI receptor protein was detected in astrocytes of normal human hypothalamic brain sections using immunocytochemical techniques. To ascertain that the cloned receptor was functional, Chinese hamster ovary (CHO) cells were transfected with a plasmid vector containing the IL-1RI coding region. IL-1RI-mediated-signal transduction was assessed by microphysiometry and activation of p38 MAP (mitogen-activated protein) kinase. We report the first demonstration that both the type I IL-1 transcript and the protein are expressed in the human hypothalamus. The receptor was expressed in a stable CHO cell line, providing a tool with which to embark on a thorough analysis of the signalling mechanisms mediated by IL-1 via this receptor.

Animals↗
PubMed1999

Relationships between learning difficulties and psychological problems in preadolescent children from a longitudinal sample.

OBJECTIVES: To examine the relationships between learning difficulties and behavior problems in preadolescent children both concurrently and longitudinally, using data from the Australian Temperament Project, and to examine associations between DSM-diagnosed disorders and types of learning difficulties. METHOD: Clinical and comparison groups of 11- to 12-year-old children were assessed on behavioral and learning indices and completed a structured diagnostic interview. Longitudinal data were available from earlier surveys of the development of these children. RESULTS: Children in the clinical group were disadvantaged on all learning and behavioral indices, and their problems in many cases had been evident from 7 to 8 years of age. There were specific associations between types of learning difficulties and both internalizing and externalizing disorders. Numeracy difficulties were strongly associated with DSM diagnoses for both boys and girls, and externalizing problems tended to co-occur with a combination of learning problems. CONCLUSIONS: Behavioral maladjustment in many domains is strongly associated with learning difficulties even after intelligence and socioeconomic factors are controlled. The odds ratios for persistence of difficulties throughout elementary school are extremely high for children with clinical problems.

Child↗
PubMed1999

Psychological disorders and their correlates in an Australian community sample of preadolescent children.

The nature and correlates of psychological disorders of preadolescent children from the longitudinal Australian Temperament Project (ATP) are reported. Almost half of the children identified via checklists completed by mothers, teachers, and the children themselves as being in the at-risk range for disorder received a DSM-III-R diagnosis. Nine per cent of comparison, or low-risk, children also received a diagnosis. Internalising disorders were the most common, and 44% of cases had multiple problems. Children rated as problematic by all three informants or by child plus teacher were the most likely to receive a diagnosis. The at-risk group had more difficult temperament, poorer family and peer relationships, lower levels of social skills, and were of lower SES than the comparison group. There were minimal differences on these variables between at-risk children who did, or did not, receive a DSM-III-R diagnosis. It is argued that especially for boys, checklists from multiple informants are effective tools for identifying clinically significant disorders.

Australia↗
PubMed1998

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices.

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

Animals↗
PubMed1998

Rat central ORL-1 receptor uncouples from adenylyl cyclase during membrane preparation.

Nociceptin/orphanin FQ is the endogenous agonist of the orphan receptor ORL-1. In this study, we sought to examine any possible regional differences of nociceptin binding using [125I]Tyr14-nociceptin, and of agonist induced inhibition of cAMP formation in membranes prepared from cerebrocortex, cerebellum and brainstem. The binding of [125I]Tyr14-nociceptin was concentration-dependent and saturable, with Bmax and pKd (pM) values of 179.7+/-15.3 fmol/mg protein and 10.26+/-0.09 (60.0), 12.4+/-1.8 fmol/mg protein and 10.44+/-0.07 (37.0), 52.3+/-0.8 fmol/mg protein and 10.16+/-0.08 (74.0) in cerebrocortical, cerebella and brainstem membranes, respectively. In all preparations, nociceptin up to 1 microM failed to inhibit basal and forskolin stimulated cAMP formation. In all tissues forskolin stimulated and nabilone (acting at the central cannabinoid receptor) inhibited cAMP formation. Collectively these data report regional differences in ORL-1 receptor expression and that these receptors uncouple during membrane preparation.

Animals↗
PubMed1998

Identification of nociceptin in human cerebrospinal fluid: comparison of levels in pain and non-pain states.

We have measured plasma and cerebrospinal fluid (CSF) concentrations of nociceptin, the endogenous agonist of the orphan opioid receptor-like receptor (ORL-1). We studied two groups of ten patients presenting for elective Caesarean section (Group E) or in established labour and requiring combined spinal epidural anaesthesia for pain relief (Group L). Nociceptin was identified in all CSF samples with mean +/- SD concentrations of 52.49 +/- 34.25 and 63.39 +/- 33.26 pg/ml in groups E and L, respectively. Nociceptin was identified in 16/20 plasma samples with mean +/- SD concentrations of 7.59 +/- 21.58 and 13 73 +/- 23.79 pg/ml in groups E and L, respectively. CSF concentrations were significantly higher than plasma concentrations and there were no differences between groups E and L. These data report the first measurements of CSF nociceptin in man and show no association with the acute pain of labour.

Adult↗
PubMed1998

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer.

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.

Animals↗
PubMed1998

Effects of C-terminal truncation of the recombinant delta-opioid receptor on phospholipase C and adenylyl cyclase coupling.

Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. We have generated two C-terminal truncation mutants of the delta-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. D-[Pen(2,5)]-enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the delta(wt), delta15, and delta37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO delta15, and 120 s in CHO delta37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose-response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating G(i alpha) coupling to adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal "strong" coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.

Adenylyl Cyclase Inhibitors↗
PubMed1997

The effects of recombinant rat mu-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase.

1. The rat mu-opioid receptor has recently been cloned yet its second messenger coupling remains unclear. The endogenous mu-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+]i and inhibits adenylyl cyclase (AC). We have examined the effects of mu-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+]i and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned mu-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+]i was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the Bmax varied between passages. Fentanyl (10 pM 1 microM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 +/- 0.1), and was best fit to a two site model, with pK1 values (% of sites) of 9.97 +/- 0.4 (27 +/- 4.8%) and 7.68 +/- 0.07 (73 +/- 4.8%). In the presence of GppNHp (100 microM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 +/- 0.1) with a pK1 value of 6.76 +/- 0.04. 4. Fentanyl (0.1 nM-1 microM) had no effect on basal, but dose-dependently inhibited forskolin (1 microM)-stimulated, cyclic AMP formation (pIC50 -7.42 +/- 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K1 = 1.7 nM). Morphine (1 microM) and [D-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 microM) also inhibited forskolin (1 microM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 microM) did not. 5. Fentanyl (0.1 nM-10 microM) caused a naloxone (1 microM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 +/- 0.15 (n-5), PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2 (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 microM) and DAMGO (1 microM), but not DPDPE (1 microM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 microM) also caused an increase in [Ca2+]i (80 +/- 16.4 nM, n-6), reaching a maximum at 26.8 +/- 2.5 s. The increase in [Ca2+]i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 microM). Pre-incubation with naloxone (1 microM, 3 min) completely abolished fentanyl-induced increases in [Ca2+]i. 6. In conclusion, the cloned mu-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+]i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous mu-opioid receptor.

Adenylyl Cyclases↗