Search PubMed⌕ Search

Biomedical subjects

C J Kirk

Publications and source records attributed to C J Kirk.

At least 55 records · Page 3Linked to original sources
PubMed1994

Simultaneous oscillations of cytoplasmic free Ca2+ concentration and Ins(1,4,5)P3 concentration in mouse pancreatic beta-cells.

Changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in stimulated cells are often oscillatory, but the mechanisms that drive these oscillations are still a matter of controversy: different models of the generation of these [Ca2+]i oscillations make different assumptions as to whether oscillations in Ins(1,4,5)P3 concentration are necessary for this process. We have looked for changes in inositol polyphosphate levels that might occur in suspensions of murine pancreatic beta-cells when these cells are induced to display synchronized oscillations in [Ca2+]i by the sequential addition of glucose, an alpha 2-adrenergic stimulus and extracellular Ca2+. The intracellular level of Ins(1,4,5)P3 oscillated in a manner approximately in synchrony with changes in [Ca2+]i. Oscillations in the levels of Ins(1,4,5)P3 metabolites [Ins(1,3,4)P3 and inositol bisphosphates] were slightly delayed relative to the Ins(1,4,5)P3 oscillations, and the concentration of Ins(1,3,4,5,6)P5 remained approximately constant during the [Ca2+]i oscillations. These results demonstrate that [Ins(1,4,5)P3] and [Ca2+]i oscillate in synchrony in at least one type of cell. Whether such oscillations in intracellular [Ins(1,4,5)P3] provide a primary driving force for [Ca2+]i oscillations either in beta-cells or in other stimulated cells remains to be determined. Even if they do not, the [Ins(1,4,5)P3] oscillations will at least provide an amplifying influence on the [Ca2+]i changes.

Animals↗
PubMed1994

In vitro neuroprotection by substituted guanidines with varying affinities for the N-methyl-D-aspartate receptor ionophore and for sigma sites.

Radioligand binding techniques were used to determine the affinity of a series of substituted guanidine derivatives for 1) the binding site within the ion channel of the N-methyl-D-aspartate (NMDA) receptor, as defined by displacement of MK-801 ([3H]dizocilpine) and 2) sigma sites as defined by displacement of [3H]N,N'-di-(o-tolyl)guanidine. The goal was to find ligands with high affinity and selectivity for the NMDA receptor ion-channel site. The neuroprotective activity of these compounds was assessed by their ability to protect cortical neurons from injury caused by a 5-min exposure to 500 microM glutamate in vitro. Release of lactate dehydrogenase into the culture medium by damaged neurons was used as an index of neuronal injury. The 14 compounds tested had IC50 values ranging from 37.3 nM to 12.7 microM for the NMDA receptor ion-channel site and from 8.3 nM to 7.25 microM for sigma sites. Affinity for the ion-channel site was improved by unsymmetrical substitutions on the guanidine moiety. All compounds in the series protected cortical neurons against glutamate toxicity, with EC50 values (concentration affording 50% protection) ranging from 0.38 to 28.25 microM. The neuroprotective effect of each compound was positively correlated with its ion-channel site affinity (r = 0.94); no correlation between neuroprotective efficacy and sigma site binding affinity was found (r V -0.13) establishing clearly that neuroprotection in this assay was linked to NMDA antagonist properties.

Animals↗
PubMed1993

Fluorescent and biotinylated linear peptides as selective bifunctional ligands for the V1a vasopressin receptor.

We have designed and synthesized a linear peptide analogue of arginine vasopressin. This peptide, [1-phenylacetyl, 2-O-methyl-D-tyrosine, 6-arginine, 8-arginine, 9-lysinamide]vasopressin (PhAcALVP), has a lysinamide residue substituted for the more usual glycinamide at position 9. Derivatization of PhAcALVP at the N epsilon-lysyl amino group with N-hydroxysuccinimide esters of aminomethylcoumarin (Mec) and biotin (Btn) produced the bifunctional ligands PhAcAL(Mec)VP and PhAcAL(Btn)VP, respectively. Pharmacological characterization of these peptides revealed that all were high-affinity V1a-selective antagonists. PhAcAL(Btn)VP can simultaneously bind to both the rat liver V1a receptor and avidin conjugates. Using this strategy, we were able to study the distribution of V1a receptors on the surface of the rat mammary tumour cell line, WRK-1. Routine epifluorescent microscopy and confocal image analysis were used to observe the distribution of avidin-Texas-Red associated with receptor-bound PhAcAL(Btn)VP. We conclude that PhAcALVP is a useful precursor for the production of hetero-bifunctional V1a-selective ligands. Both PhAcAL(Mec)VP and PheAcAL(Btn)VP can be used selectively to probe the V1a receptor and will be versatile tools for a variety of histocytochemical applications, including receptor localization and purification.

Amino Acid Sequence↗
PubMed1993

Helicopter Emergency Medical Service operating from the Royal London Hospital: the first year.

The additional workload on the support hospital created by using a helicopter for emergency trauma has been assessed in the first year of operation. A total of 135 patients were brought to the Royal London Hospital, of whom 30 died, 34 were transferred for convalescence or rehabilitation to another hospital, and 71 discharged home. The median Injury Severity Score was 17 (range 0-75) and 82 patients (60.7 per cent) had a reduced Revised Trauma Score at the scene. Blunt injury greatly outnumbered penetrating trauma (117 versus 15 patients). Accidental injuries accounted for 77.0 per cent of the total, self-inflicted wounds 14.1 per cent and assaults 8.9 per cent. Parameters employed to assess workload were hospital bed-days used (total 2361), operations (118, occupying 287 h of theatre time) and blood transfusion (total 702 units used). The second 6-month study period showed a considerable increase in the number of patients admitted (102 versus 33) because of increased efficiency in call-out procedures.

Adolescent↗
PubMed1992

The inositol phosphates in WRK1 rat mammary tumour cells.

1. A detailed structural survey has been made of the inositol phosphates of unstimulated and vasopressin-stimulated WRK-1 rat mammary tumour cells. Inositol phosphate peaks were separated by h.p.l.c., and structural assignments were made for more than 20 compounds by combinations of: (a) co-chromatography with labelled standards; (b) site-specific enzymic dephosphorylation; (c) complete and partial periodate oxidation, followed by h.p.l.c. of polyols and their stereospecific oxidation by dehydrogenases; and (d) ammoniacal hydrolysis. 2. The 'inositol monophosphates' fraction from unstimulated cells included an uncharacterized peak, probably containing some glycerophosphoinositol, and Ins(1:2-cyclic)P. Stimulation provoked accumulation of both Ins1P and Ins3P, of Ins2P, and of Ins5P and/or the enantiomers Ins4P and Ins6P. The proportions of Ins1P and Ins3P were determined by partial periodate oxidation and enantiomeric identification of the resulting glucitols. 3. Three inositol bisphosphate peaks were detected in unstimulated cells: Ins(1,4)P2 [this was distinguished chemically from its enantiomer Ins(3,6)P2], Ins(3,4)P2 and/or Ins(1,6)P2, and Ins(4,5)P2 and/or Ins(5,6)P2. On stimulation, Ins(1,4)P2 and Ins(3,4)P2 [and/or Ins(1,6)P2] levels increased, and Ins(1:2-cyclic,4)P2 and Ins(1,3)P2 were also formed. 4. Three inositol trisphosphate peaks were obtained from unstimulated cells: all increased during stimulation. These were Ins(1,3,4)P3 [with some Ins(1:2-cyclic,4,5)P3], Ins(1,4,5)P3 and Ins(3,4,5)P3 [and/or Ins(1,5,6)P3]. During stimulation, another compound, probably Ins(1,4,6)P3, appeared in the 'Ins(1,4,5)P3 peak'. The 'Ins(3,4,5)P3 peak' contained a second trisphosphate, probably Ins(2,4,5)P3. 5. Three inositol tetrakisphosphates, namely Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, were present in unstimulated cells, and all accumulated during stimulation. 6. Ins(1,3,4,5,6)P5, which is the most abundant inositol polyphosphate in these cells, a less abundant inositol pentakisphosphate and inositol hexakisphosphate were all unresponsive to stimulation.

Animals↗
PubMed1992

The interrelationships of the inositol phosphates formed in vasopressin-stimulated WRK-1 rat mammary tumour cells.

1. Temporal changes in the levels of many inositol phosphates, whose structural characterization is presented in the preceding paper [Wong, Barker, Morris, Craxton, Kirk & Michell (1991) Biochem. J. 286, 459-468], have been monitored in vasopressin-stimulated WRK-1 cells. 2. Upon stimulation, Ins(1,4,5)P3 accumulated within 1 s, consistent with its role as a rapidly acting second messenger produced by receptor activation of phosphoinositidase C. Ins(1,4)P2 and Ins(1,3,4,5)P4, both of which are immediate products of Ins(1,4,5)P3 metabolism, also accumulated quickly. Ins4P, Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2, Ins1P and Ins3P, which are intermediates in the metabolism of Ins(1,4)P2 and Ins(1,3,4,5)P4 to inositol, accumulated after seconds or within a few minutes, and in a temporal sequence consistent with their known metabolic interrelationships. 3. The stimulated accumulation of Ins(1,3,4,6)P4 was delayed, as expected if it is formed by phosphorylation of Ins(1,3,4)P3. 4. Ins(3,4,5,6)P4 accumulated 2-3-fold in a few minutes, and mainly before Ins(1,3,4,6)P4. 5. Using a [3H]-/[14C]-inositol double-labelling protocol, we obtained evidence that all of the compounds that accumulated upon stimulation, except Ins(3,4,5,6)P4, originated from lipid-derived Ins(1,4,5)P3, but that the newly formed Ins(3,4,5,6)P4 came from a different source. 6. There were no consistent changes in the levels of Ins(1,3,4,5,6)P5 and InsP6 during stimulation. 7. Alongside the gradual accumulation of Ins(1:2-cyclic,4,5)P3 during stimulation [Wong, Barker, Shears, Kirk & Michell (1988) Biochem. J. 252, 1-5], there was an accumulation of Ins(1:2-cyclic,4)P2 and Ins(1:2-cyclic)P, probably as either minor side products of phosphoinositidase C action or metabolites of Ins(1:2-cyclic,4,5)P3. 8. When Li+ was present during stimulation, it redirected the dephosphorylation pathways downstream of Ins(1,4,5)P3 in the manner expected from its inhibition of inositol monophosphatase and Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase: there were marked increases in the accumulation of Ins(1,4)P2 and Ins(1,3,4)P3 and of monophosphates. Moreover, Li+ shifted the Ins1P/Ins3P balance in favour of Ins1P, thus demonstrating redirection of the metabolism of the accumulated Ins(1,3,4)P3 towards Ins(1,3)P2 rather than Ins(3,4)P2.

Animals↗
PubMed1992

The opening of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel in rat cerebellum is inhibited by caffeine.

Ins(1,4,5)P3(InsP3)-induced Ca2+ release and [3H]InsP3 binding were measured in rat cerebellar microsomes in the presence or absence of caffeine. The quantal Ca2+ release was shown to occur in an apparently co-operative fashion with a Hill coefficient (h) of 2.2. Half-maximal Ca2+ release was observed at 900 nM-InsP3. Addition of caffeine caused changes both to the concentration of InsP3 required to cause half-maximal Ca2+ release (3.9 microM at 50 mM-caffeine) and to the apparent co-operativity (h = 1.0 at 50 mM-caffeine). Under standard conditions for [3H]InsP3 binding, caffeine had no effect, and it had no effect on InsP3 metabolism. Cyclic AMP also had no effect on the quantal release induced by InsP3. These results are consistent with the view that caffeine affects the opening (Ca2+ release) events rather than the ligand-binding events in the operation of the InsP3-sensitive Ca2+ channel.

Adenosine Triphosphate↗
PubMed1992

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.

Abortion, Spontaneous↗
PubMed1991

Characterization of the human liver vasopressin receptor. Profound differences between human and rat vasopressin-receptor-mediated responses suggest only a minor role for vasopressin in regulating human hepatic function.

The [Arg8]vasopressin (AVP) receptor expressed by human hepatocytes was characterized, and compared with the rat hepatic V1a vasopressin receptor subtype. In addition to determining the pharmacological profile of the human receptor, the cellular responses to AVP were measured in human and rat hepatocytes by assaying glycogen phosphorylase alpha activity and DNA synthesis. Marked differences were observed between human and rat hepatocytes regarding vasopressin receptors and the intracellular consequences of stimulation by AVP. Data presented in this paper demonstrate the following, (i) Vasopressin V1a receptors are present in low abundance on human hepatocytes. (ii) Species differences exist between human and rat V1a receptors with respect to the affinity of some selective antagonists. (iii) AVP-stimulated glycogen phosphorylase a activation in human hepatocytes was approx. 5% of that observed in rat cells. (iv) In contrast with rat hepatocytes, DNA synthesis in human cells in culture was not stimulated by AVP. It is concluded that vasopressin plays only a minor role in the regulation of human hepatic function. Furthermore, conclusions drawn from observations made with AVP and its analogues on rat hepatic function cannot be directly extrapolated to the human situation.

Animals↗
PubMed1991

A salt-activated inositol 1,3,4,5-tetrakisphosphate 3-phosphatase at the inner surface of the human erythrocyte membrane.

The localization of the human erythrocyte membrane Ins(1,3,4,5)P4 3-phosphatase was investigated by saponin permeabilization of resealed 'isoionic' erythrocyte ghosts. This enzyme is active at the inner face of the plasma membrane, at the same site as a specific 5-phosphatase that degrades both Ins (1,4,5)P3 and Ins(1,3,4,5)P4. In the presence of EDTA, Ins(1,4,5)P3 was the only product of Ins(1,3,4,5)P4 metabolism. However, when Mg2+ was present both the 5-phosphatase and the 3-phosphatase attacked Ins (1,3,4,5)P4, directly forming Ins(1,3,4)P3 and Ins(1,4,5)P3;some Ins(1,4)P2 was also formed as a product of 5-phosphatase attack on the liberated Ins(1,4,5)P3. The Ins(1,3,4,5)P4 3-phosphatase was potently activated by KCl, thus making the route of metabolism of Ins(1,3,4,5)P4 by erythrocyte ghosts strikingly sensitive to variations in ionic strength: at 'cytosolic' K+ and Mg2+ levels, 3-phosphatase activity slightly predominated over 5-phosphatase. Ins(1,3,4,5)P4 3-phosphatase was potently inhibited by Ins-(1,3,4,5,6)P5 and InsP6 at levels lower than those often observed within cells. This leaves open the question as to whether the cellular function of inositol polyphosphate 3-phosphatase is to participate in a physiological cycle that interconverts Ins(1,3,4,5)P4 and Ins(1,4,5)P3 or to metabolize other inositol polyphosphates in the cytosol compartment of cells.

Erythrocyte Membrane↗
PubMed1990

Inositol phosphates in receptor-mediated cell signaling: metabolic origins and interrelationships.

We have investigated the metabolic interrelationships of the major inositol phosphates in vasopressin-stimulated WRK 1 mammary tumor cells which were labeled to equilibrium with [14C]inositol and briefly, just prior to stimulation, with [3H]inositol. A comparison of the 3H/14C ratios of these compounds with those of the cellular inositol lipids suggests that most of the known inositol mono-, bis-, tris-, and tetrakis-phosphates are derived from precursors with turnover rates similar to those of these lipids. However, Ins(3,4,5,6)P4 (which is the major inositol tetrakisphosphate to accumulate in stimulated WRK 1 cells), Ins(1,3,4,5,6)P5, and InsP6 had 3H/14C ratios of 0 in this experiment, indicating that they must have a different metabolic origin.

Animals↗