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Biomedical subjects

S P Watson

Publications and source records attributed to S P Watson.

At least 19 recordsLinked to original sources
PubMed1992

Tumor necrosis factor alpha stimulates sphingomyelinase through the 55 kDa receptor in HL-60 cells.

Tumor necrosis factor alpha (TNF alpha) stimulated rapid (seconds) hydrolysis of sphingomyelin in HL-60 cells, formation of phosphocholine (PCho) and a decrease in choline. The response to TNF alpha was concentration dependent with a maximal effect at 3-10 nM. The monoclonal antibody (mAb), htr-9, which behaves as an agonist at the 55 kDa subtype of the TNF receptor, also stimulated sphingomyelin hydrolysis in intact cells. In contrast, the mAb, utr-1, which behaves as an antagonist at the 75 kDa receptor subtype, had no effect on sphingomyelin hydrolysis either on its own or in the presence of TNF alpha. In addition, htr-9 or TNF alpha stimulated hydrolysis of sphingomyelin in a membrane fraction of HL-60 cells. These results are consistent with a role of sphingomyelin hydrolysis as an early event in the signalling mechanism of TNF alpha, and suggest that this pathway is activated through the 55 kDa subtype of the TNF receptor.

Antibodies, Monoclonal↗
PubMed1992

Okadaic acid inhibits activation of phospholipase C in human platelets by mimicking the actions of protein kinases A and C.

1. The effect of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), on human platelets has been investigated. 2. Okadaic acid exerts a general increase in phosphorylation of platelet proteins but did not induce aggregation or secretion of 5-hydroxytryptamine (5-HT). Okadaic acid, however, did inhibit thrombin-induced functional responses. 3. Maximally effective concentrations of prostacyclin, to elevate adenosine 3'-5'-cyclic monophosphate (cyclic AMP), or phorbol dibutyrate, to activate protein kinase C, inhibited the formation of inositol phosphates by thrombin by approximately 60%. When used in combination, prostacyclin and phorbol dibutyrate reduced the levels of inositol phosphates induced by thrombin to 11%. 4. Okadaic acid (1 microM) decreased thrombin-induced formation of inositol phosphates by approximately 55% and increased the inhibitory action of prostacyclin or phorbol dibutyrate. Okadaic acid had no further effect when prostacyclin and phorbol dibutyrate were used in combination. 5. These results suggest that protein kinases A and C act to inhibit phospholipase C by distinct mechanisms and that their action is reversed by PP1 and/or PP2A.

Blood Platelets↗
PubMed1992

Chronic lithium treatment inhibits basal and agonist-stimulated responses in rat cerebral cortex and GH3 pituitary cells.

Li+ is used clinically in the management of bipolar-disordered (manic-depressive) illness, but the mechanism of its clinical efficacy remains unclear. Li+ inhibits the metabolism of certain inositol phosphates, leading to a decreased cycling of inositol that may be sufficient to reduce phosphoinositide metabolism. We have tested this hypothesis in slices of rat cerebral cortex and in rat pituitary GH3 cells grown in the presence of low extracellular inositol. We show that basal and stimulated mass levels of inositol-1,4,5-trisphosphate were reduced in rat cerebral cortex and in GH3 cells after chronic, but not acute, treatment with a therapeutic concentration of Li+. In GH3 cells chronic treatment with Li+ also decreased basal levels of intracellular Ca2+ and secretion of prolactin, effects that were prevented by the presence of myo-inositol. Agonist-stimulated mobilization of Ca2+ and prolactin release were also reduced in Li(+)-treated cells. These findings show that chronic perturbation of the phosphoinositide pathway by Li+ is sufficient to reduce basal and agonist-stimulated cellular responses, an action that may underlie its effectiveness in the alleviation of affective disorders.

Animals↗
PubMed1991

The presence of NK3 tachykinin receptors on rat uterus.

The NK3 agonist, senktide, induced a potent contraction of rat uterus in the presence of tetrodotoxin, atropine and indomethacin, or the tachykinin receptor antagonists L-659877 and [D-Pro4,D-Trp7,9,10]substance P (4-11). Additional contractile and radioligand binding studies with receptor selective agonists and antagonists confirmed the presence of NK3 receptors and also revealed the presence of NK1 and NK2 receptors. The rat uterus is the second peripheral tissue in which a post-synaptic, non-neuronal NK3 receptor has been identified.

Animals↗
PubMed1991

Influence of Mg2+ and pH on n.m.r. spectra and radioligand binding of inositol 1,4,5-trisphosphate.

We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3.

Adrenal Cortex↗
PubMed1990

Are there subtypes of the inositol 1,4,5-trisphosphate receptor?

We have compared the properties of the [3H]Ins(1,4,5)P3-binding sites from a number of tissues in an attempt to determine if heterogeneity exists within the Ins(1,4,5)P3-receptor family. The binding of Ins(1,4,5)P3 was characterized in detail by using membranes prepared from human uterine smooth muscle and bovine adrenal cortex. Ins(1,4,5)P3 exhibited an approx. 5 times greater affinity for the binding site in adrenal cortex (KD = 9.81 +/- 1.92 nM) compared with uterine smooth muscle (KD = 37.1 +/- 1.8 nM). The binding was dependent on pH in both tissues, with a maximum at pH 8.3; at this pH various inositol phosphates and nucleotides competed for the binding sites with similar potencies on both tissues. However, the binding of Ins(1,4,5)P3 to the uterine smooth-muscle membranes was Ca2(+)-sensitive, whereas that to the bovine adrenal cortex was not; furthermore, heparin displaced the binding of Ins(1,4,5)P3 in the uterus with an IC50 value (concn. of displacer giving 50% inhibition of specific binding) of 3.9 micrograms/ml (2.5, 6.4; lower, upper range), compared with a value of 22 (13, 30) micrograms/ml in adrenal cortex. In view of the ability of Ins(1,4,5)P3 and heparin to distinguish between these binding sites, their effect on other tissues was examined. Ins(1,4,5)P3 showed a similar affinity for receptors located in the bovine cerebellum to those in the bovine adrenal cortex, but heparin displaced Ins(1,4,5)P3 binding with a 5-fold greater affinity from the cerebellum. Ins(1,4,5)P3 had a 2-fold greater affinity for its receptor with human platelets, as compared with human uterus, but heparin was unable to distinguish between these sites. In guinea-pig ileum, Ins(1,4,5)P3 displayed a similar affinity for the receptors in the longitudinal muscle compared with the circular muscle, but heparin could distinguish between these sites. These data show that small differences exist between tissues, but no clear picture is apparent. It is possible that these results reflect tissue-dependent factors such as phosphorylation, the presence of calmedin etc., rather than the presence of receptor subtypes or species difference.

Adrenal Cortex↗
PubMed1990

Lithium potentiates agonist formation of [3H]CDP-diacylglycerol in human platelets.

Thrombin stimulated a rapid formation of [3H]CDP-diacylglycerol in platelets prelabelled with [3H]cytidine. This response was increased in the presence of LiCl after a delay of 5 min; potentiation could be prevented by myo-inositol. Since Li+ inhibits the liberation of inositol from inositol phosphates, the conversion of diacylglycerol to phosphatidylinositol via CDP-diacylglycerol may be dependent on the regeneration of inositol from this pathway.

Blood Platelets↗
PubMed1990

Pharmacological analysis of [3H]-senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes.

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive. 5. The binding of [3H]-senktide to cortex membranes was inhibited by GTP (p1C,0 = 6.49)and GTP-gamma- S (p1C,0 = 6.67) with ATP being at least three orders of magnitude less potent (pIC50 = 3.55). 6. These results indicate that both central and peripheral NK3 receptors share a similar pharmacological specificity and that they may be labelled selectively with the NK3 receptor agonist [3H]-senktide.

Animals↗
PubMed1990

K(+)-stimulation of the phosphoinositide pathway in guinea-pig ileum longitudinal smooth muscle is predominantly neuronal in origin and mediated by the entry of extracellular Ca2+.

1. K+ and scorpion toxin stimulate formation of inositol phosphates in guinea-pig ileum longitudinal smooth muscle slices. The response to these two agents is not additive. 2. The response to K+ is inhibited partially by nifedipine and partially by omega-conotoxin. When given together the effect of these two Ca2+ channel blockers is additive and the response to K+ is reduced by more than 80%. 3. The response to scorpion toxin is inhibited completely by tetrodotoxin, partially by omega-conotoxin but not by atropine or nifedipine. Scorpion toxin induces a similar formation of inositol phosphates in collagenase-dispersed cells to that seen in cross-chopped slices. 4. The responses to scorpion toxin and K+ are inhibited completely when the extracellular Ca2+ concentration is reduced to below cytosolic levels (less than 100 nM). 5. Neither nifedipine nor omega-conotoxin, either alone or in combination, inhibited formation of inositol phosphates by substance P or carbachol. Both of these agonists induced a significant formation of inositol phosphates even when the extracellular Ca2+ concentration was reduced to 10 nM. 6. These results indicate that K+ and scorpion toxin induce formation of inositol phosphates through the mobilisation of extracellular Ca2+. The response to K+ appears to occur predominantly in neuronal cells.

Animals↗
PubMed1990

Phorbol esters inhibit smooth muscle contractions through activation of Na(+)-K(+)-ATPase.

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.

Animals↗
PubMed1990

Inositol 1,4,5-trisphosphate and oxytocin binding in human myometrium.

The presence of inositol 1,4,5-trisphosphate (IP3) receptors in human myometrium has been investigated and their concentration compared with that of oxytocin receptors. Myometrial microsomes were incubated with 3H-IP3 alone and in the presence of unlabeled IP3. Binding was to a single class of noninteracting sites with a density of 1-2 pmol/mg of protein. The sites had characteristics of true IP3 receptors, i.e., very fast association and dissociation rates, high affinity (Kd 25-50 nM) and specificity (IP3 greater than IP3[2,4,5], IP4 much greater than IP5 greater than IP3[1,3,4], IP1, IP2, IP6), and did not metabolize 3H-IP3. The binding was maximal at pH 8, and was inhibited by calcium (IC50 = 80 nM), magnesium (IC50 = 100 microM), heparin (IC50 = 4.5 micrograms/ml), and GTP (IC50 = 150 microM). The concentration and affinity of IP3 receptors were similar in pregnant and nonpregnant myometrium and remained constant during labor. By contrast, the density of oxytocin receptor increased significantly from nonpregnant to pregnant tissue and fell in advanced spontaneous labor but not in advanced induced labor. These results provide new, additional evidence for the involvement of the phosphatidylinositol pathway in the control of uterine contractility.

Binding, Competitive↗
PubMed1989

Phosphorylation-dependent and -independent pathways of platelet aggregation.

We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

Alkaloids↗
PubMed1989

Protein kinase C regulates the tonic but not the phasic component of contraction in guinea-pig ileum.

1. We have investigated the effect of phorbol esters and the down-regulation of protein kinase C on contraction of guinea-pig ileum longitudinal smooth muscle to carbachol and high K+. 2. Phorbol 12,13-dibutyrate (PDBu) enhanced the phasic component and inhibited or enhanced, respectively, the tonic component of contraction to carbachol and high K+. In contrast, 4 alpha-phorbol, which does not activate protein kinase C, had no effect on these responses. 3. Exposure to phorbol 12-myristate 13-acetate (PMA; 1 microM) for up to 8 h induced a time-dependent loss of [3H]-PDBu binding sites, consistent with the down-regulation of protein kinase C by this treatment. 4. The phasic component of contraction to carbachol or high K+ was unaffected following the down-regulation of protein kinase C. The tonic component of contraction to carbachol was markedly enhanced by this treatment while that to high K+ was partially suppressed. 5. These data suggest that although the activation of protein kinase C can lead to potentiation of the phasic component of contraction to carbachol or high K+, this appears to have little physiological significance since the response is not altered in tissues in which protein kinase C has been down-regulated. On the other hand, protein kinase C may limit the tonic contraction to carbachol but potentiate that to high K+.

Animals↗
PubMed1988

Fluoride inhibits agonist-induced formation of inositol phosphates in rat cortex.

Sodium fluoride inhibited carbachol, 5-hydroxytryptamine and noradrenaline stimulated formation of inositol phosphates in rat cerebral cortex. For example, carbachol (1 mM) induced a 337% increase of inositol phosphates above basal in 30 min which was reduced to 69% in the presence of NaF (10 mM). The IC50 for NaF was approximately 1.5 mM and inhibition was mediated by a decrease in maxima of the carbachol dose response curve rather than a shift to the right. This inhibitory action was not mimicked by NaBr or NaI, or by agents which increase cAMP. Inhibition did not appear to result from a toxic action of NaF since it had no effect on the formation of inositol phosphates by high K+; moreover, in higher concentrations NaF stimulated phospholipase C activity. Since fluoride ions are known to activate G-proteins in the concentrations used in this study, these results may indicate the existence of a novel G-protein linked to receptor inhibition of phospholipase C.

Animals↗
PubMed1988

Lowering of the extracellular Na+ concentration enhances high-K+-induced formation of inositol phosphates in the guinea-pig ileum.

1. Formation of inositol phosphates (InsPs) was measured in cross-chopped slices or dispersed cells, isolated by collagenase treatment, of guinea-pig ileum longitudinal smooth muscle pre-labelled with [3H]inositol. 2. Elevation of the extracellular K+ concentration by equimolar replacement of Na+ induced accumulation of InsPs in the dispersed cells and in the tissue slices. These effects were blocked by neither tetrodotoxin (1 microM) nor atropine (10 microM), and were approximately additive with carbachol-induced accumulation. 3. In the tissue slices, the response to K+ was partially inhibited by nifedipine (10 microM) and by CdCl2 (0.3 mM), but the carbachol-induced response was not altered. 4. Accumulation of InsPs induced by KCl-excess solution (high-K+ solution without Na+ replacement) was suppressed strongly by nifedipine and completely by CdCl2. The response to KCl excess was approx. 40% of that to high K+ with Na+ replacement. 5. Low-NaCl solution (replacement of NaCl with equimolar sucrose) also produced InsPs, and this was not blocked by either nifedipine (10 microM) or CdCl2 (0.3 mM). 6. The formation of InsPs by a maximally effective concentration of carbachol (1 mM) in the presence of KCl excess or low NaCl was greater than the additive effect of the two stimuli on their own. Enhancement of the carbachol-induced response by KCl excess disappeared in the presence of CdCl2 (0.3 mM). 7. These data suggest that formation of InsPs induced by high-K+ solution with equimolar replacement of Na+ consists of two components, i.e. high-K+-induced inositol-phospholipid hydrolysis by Ca2+ entry through voltage-sensitive channels, and low-Na+-induced formation of InsPs, insensitive to Ca2+ antagonists, but that both of them do not contribute significantly to the activation of phospholipase C by muscarinic stimuli.

Animals↗