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C J Kirk

Publications and source records attributed to C J Kirk.

At least 19 recordsLinked to original sources
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↗
PubMed1989

Influence of bacterial toxins and forskolin upon vasopressin-induced inositol phosphate accumulation in WRK 1 cells.

The accumulation of inositol phosphates in WRK 1 cells, stimulated with a range of vasopressin concentrations, was diminished by prior exposure to cholera toxin or forskolin, whilst that observed in the presence of maximal concentrations of the hormone was enhanced in pertussis-toxin-treated cells. In the presence of [32P]NAD+, both cholera toxin and pertussis toxin provoked the labelling of peptides with approximate Mrs of 45,000 and 41,000 respectively in the membranes of WRK 1 cells. Exposure to cholera toxin or forskolin for 15-18 h enhanced cyclic AMP accumulation in these cells. The concentrations of these agents which provoked half-maximal cyclic AMP accumulation were similar to those required to diminish receptor-mediated inositol phosphate accumulation by 50%. In contrast, half-maximal ADP-ribosylation of the 45,000Mr peptide needed 100-fold greater concentrations of the toxin than were effective in provoking half-maximal inhibition of inositol phosphate accumulation. Cholera toxin or forskolin also reduced the maximal specific binding, to intact WRK 1 cells, of both [3H][Arg8]vasopressin and the V1a antagonist [3H][beta-mercapto-beta,beta-cyclopentamethylenepropionic acid,O-methyl-Tyr2, Arg8]vasopressin. The kinetics for the loss of this binding capacity following cholera-toxin treatment were very similar to those describing the diminution of vasopressin-stimulated inositol phosphate accumulation in the same cells.

Adenosine Diphosphate↗
PubMed1989

Non classical, multiple-site interaction of [3H]-prazosin with the alpha 1-adrenoceptor of intact BC3H1 cells.

1. In intact BC3H1 cells the EC50 of noradrenaline (NA) for the inositol phosphate response measured at 37 degrees C (EC50 = 193 nM) was much lower than its apparent dissociation constant (Ki37 degrees C = 83.211 microM) determined at this temperature by [3H]-prazosin binding. 2. After pretreatment of the cells with NA at 37 degrees C for 45 min, the time used in binding assays at this temperature, this difference between EC50 and Ki37 degrees C did not decrease significantly. An agonist-induced reduction in alpha 1-adrenoceptor affinity can therefore not explain the very high Ki37 degrees C value. 3. NA pretreatment at 37 degrees C decreased the number of [3H]-prazosin binding sites (assessed by whole cell binding at 2 degrees C) by only 49%; not by 100%, the value expected if agonist-induced receptor internalization were the origin of the very low Ki37 degrees C. 4. The EC50 of NA for the inositol phosphate response in the presence of 156 pM [3H]-prazosin was 1.841 microM but the IC50 of NA for the inhibition of [3H]-prazosin binding (126 pM) was 316 microM. As there is no alpha 1-adrenoceptor reserve in these cells we propose that at 37 degrees C [3H]-prazosin interacts, not only with the catecholamine recognition site (site 1) of the receptor, but also reacts weakly with another site from which it cannot be directly displaced by catecholamine-like substances (site 2).

Binding, Competitive↗
PubMed1989

The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover.

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [14C]inositol and then supplying them with [3H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively 3H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li+). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F2 alpha, have largely failed to demonstrate the formation of relatively 3H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher 3H: 14C ratios than phosphatidylinositol, but the 3H: 14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the 3H: 14C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.

Animals↗
PubMed1988

Preferential localization of rat liver D-myo-inositol 1,4,5-trisphosphate/1,3,4,5-tetrakisphosphate 5-phosphatase in bile-canalicular plasma membrane and 'late' endosomal vesicles.

Previous studies have shown that most of the inositol 1,4,5-trisphosphate/inositol 1,3,4,5-tetrakisphosphate 5-phosphatase activity of rat hepatocytes is associated with the plasma membrane [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. We now show that the specific activity of this enzyme is highest in the bile-canalicular domain of the plasma membrane, at the opposite pole of the hepatocyte from the presumed site of receptor-mediated formation of inositol 1,4,5-trisphosphate. In intact hepatocytes and in sealed membrane vesicles originating from the bile-canalicular domain of the plasma membrane, the 5-phosphatase activity was mostly latent and therefore located at the cytoplasmic surface. A substantial amount of 5-phosphatase was also found in rat liver endosomal fractions, particularly a 'late' endosomal subfraction, indicating that this enzyme may be transported between the sinusoidal plasma membrane and other cellular membranes.

Animals↗
PubMed1988

Inositol lipids: receptor-stimulated hydrolysis and cellular lipid pools.

Our current knowledge of the process by which receptors stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has its origin in the discovery by Hokin & Hokin (J. biol. Chem. 263, 967 (1953] that some pancreatic secretagogues not only elicit exocrine secretion but also stimulate the metabolism of membrane phospholipids. Despite the recent elucidation of many aspects of this widespread signalling system, there is still little information on the control of the supply of its substrate, PtdIns(4,5)P2. In particular, some studies have suggested that inositol-lipid-mediated signalling involves much or all of the inositol lipid complement of the stimulated cells, whereas other observations have equally clearly implicated the receptor-activated hydrolysis of an inositol phospholipid pool that comprises only a small fraction of the total cellular complement of these lipids. These studies, which have largely employed radiochemical analyses using single isotopes, are briefly reviewed. In addition, we report the first information obtained by a new procedure for analysing the metabolic characteristics of the inositol lipids that are broken down during stimulation. This technique employs cells that are doubly labelled in the inositol moiety of their lipids (to isotopic equilibrium with 14C and only briefly with 3H) to search for functional metabolic heterogeneity among the inositol lipids of stimulated cells. Using this method, we have found that the inositol phosphates liberated in stimulated cells during brief stimulation of V1a-vasopressin receptors or prostaglandin F2 alpha receptors come from phospholipid that has a turnover rate typical of the bulk of the cellular inositol lipids.

Animals↗
PubMed1988

Inositol 1:2(cyclic),4,5-trisphosphate is not a major product of inositol phospholipid metabolism in vasopressin-stimulated WRK1 cells.

1. A method has been devised for quenching cell incubations with an aqueous phenol/chloroform/EDTA mixture of neutral pH, to allow the analysis of acid-labile cell components. 2. Using this method, we have searched for the appearance of Ins(1:2cyclic,4,5)P3 [inositol 1:2(cyclic),4,5-trisphosphate] in WRK1 mammary tumour cells that were labelled to high specific radioactivity with [3H]inositol and then stimulated with 0.4 microM-vasopressin. 3. Vasopressin caused a very rapid accumulation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate), followed by a slower decline towards the original concentration. An acid-labile and inositol-labelled compound with the chromatographic properties of Ins(1:2cyclic,4,5)P3 was present in unstimulated cells at less than 5% of the elevated concentration of Ins(1,4,5)P3. Its concentration rose 2-3-fold during stimulation for 3 min, at which time its concentration was about 5% of the elevated concentration of Ins(1,4,5)P3. 4. We conclude that Ins(1,4,5)P3 is the major product of phosphoinositidase C-catalysed phosphatidylinositol 4,5-bisphosphate hydrolysis in vasopressin-stimulated WRK1 cells. Ins(1:2cyclic,4,5)P3 is unlikely to be an important intracellular messenger in these cells, at least during the first few minutes of stimulation.

Arginine Vasopressin↗
PubMed1988

Complex interactions of agonists with alpha 1-adrenoceptors in intact cells.

1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

Adrenergic alpha-Agonists↗