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Biomedical subjects

M F Crouch

Publications and source records attributed to M F Crouch.

46 records · Page 3Linked to original sources
PubMed1989

Relationship of inositol phospholipid metabolism to phospholipase A2.

The mechanism by which agonists stimulate phospholipase A2 of platelets is still much of a mystery. We have presented a discussion that suggests that neither Ca2+, protein kinase C or dissociation of the inhibitory GTP-binding protein Gi is solely responsible for activating this enzyme. We cannot exclude the possibility that there may be some contribution of each pathway for some agonists, and that the contribution may change with agonist concentration or potency. These possibilities await further clarification.

Blood Platelets↗
PubMed1988

Spacial isolation of protein kinase C activation in thrombin stimulated human platelets.

Thrombin stimulation of human platelets is associated with turnover of inositol phospholipids, mobilization of intracellular Ca2+ stores, and activation of protein kinase C. However, within 5 minutes, the thrombin receptor desensitizes, but can be re-coupled to its effectors by stimulation of alpha 2-adrenergic receptors (Crouch and Lapetina, J. Biol. Chem. 263, 3363-3371, 1988). This effect of epinephrine was found to be inhibited by preincubation of platelets with phorbol ester, suggesting that protein kinase C was inhibitory. However, since thrombin also activated protein kinase C and epinephrine was active following thrombin stimulation of platelets, this implied that thrombin activation of protein kinase C may have been spacially isolated near the thrombin receptor and could not inactivate alpha 2-receptor activity. In the present paper, we have tested this possibility, and we present evidence which strongly favours the possibility that protein kinase C activation by receptors induces its local translocation to the cell membrane.

Blood Platelets↗
PubMed1988

No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation.

Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.

Adenosine Diphosphate Ribose↗
PubMed1988

A role for Gi in control of thrombin receptor-phospholipase C coupling in human platelets.

Stimulation of washed human platelets with alpha-thrombin was accompanied by aggregation, formation of inositol phosphates and phosphatidic acid, liberation of arachidonic acid, mobilization of intracellular Ca2+ stores, and influx of Ca2+ from the extracellular medium. Each of these responses was potentiated by a short pretreatment with epinephrine, although alone this agent was ineffective. A prolonged (5 min) stimulation with alpha-thrombin desensitized both phospholipase C and Ca2+ mobilization to a further thrombin challenge. Epinephrine added following thrombin desensitization restored both the ability of thrombin to release Ca2+ stores and stimulate inositol phospholipid hydrolysis. Resensitization was mediated by alpha 2-adrenergic receptors and lasted about 3 min, after which the Ca2+ levels returned again to basal levels. Pretreatment of platelets with phorbol dibutyrate at concentrations which specifically activate protein kinase C increased the rate of desensitization of the thrombin-induced release of Ca2+ stores and abolished the ability of epinephrine to restore the thrombin response. The protein kinase C inhibitor, staurosporine, blocked the inhibitory effect of phorbol ester and also reduced the rate of desensitization of thrombin and subsequent epinephrine action. These results suggest that thrombin activation of protein kinase C phosphorylates and inactivates a signaling protein which is common to both thrombin and alpha 2-adrenergic receptors. This protein is involved in thrombin stimulation of phospholipase C but is not directly stimulatory since epinephrine alone does not activate this enzyme. We searched for a known second messenger protein common to both thrombin and alpha 2-adrenergic receptors which was phosphorylated in intact platelets by protein kinase C in parallel with thrombin-induced desensitization. The alpha subunit of the inhibitory GTP-binding protein, Gi, was the only candidate which fulfilled all of these criteria as shown by immunoprecipitation. Therefore, we suggest that alpha i maintains the thrombin receptor in a state which can couple to phospholipase C when activated with thrombin. This permissive state of alpha i is blocked by phosphorylation by thrombin-activated protein kinase C.

Arachidonic Acid↗
PubMed1988

The Na+/H+ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine.

Stimulation of platelets with thrombin, ADP and epinephrine has recently been shown to activate a Na+/H+ antiporter, with a resulting alkalinization of the cytoplasm. Unlike thrombin, however, epinephrine is incapable of directly activating phospholipase C, but is well known to potentiate the effects of thrombin on this enzyme and other subsequent steps of platelet activation. Therefore, we have studied the involvement of the Na+/H+ antiporter in this aspect of epinephrine action to see whether alkalinization of platelet cytosol could be a requirement for agonists to stimulate inositol phospholipid hydrolysis and mobilize cellular Ca2+ stores. Alpha-thrombin induced the rapid formation of inositol trisphosphate with a parallel mobilization of intracellular Ca2+ stores. Epinephrine alone had no effect on either of these parameters. The response to thrombin desensitized over a period of minutes, and after this had occurred, epinephrine was able to activate phospholipase C and induce the release of intracellular Ca2+. This showed that epinephrine was able to recouple thrombin receptors to phospholipase C, and this appeared to be mediated by the same mechanism which is involved in potentiation by epinephrine of thrombin-stimulation of phospholipase C. These effects of epinephrine were not altered by inhibition of the Na+/H+ antiporter with ethylisopropylamiloride or by use of the Na+/H+ ionophore, monensin. We conclude that epinephrine potentiates thrombin-induced responses by a mechanism which is unrelated to its effects on the Na+/H+ antiporter, and this is not a requirement for thrombin-induced phospholipase C activation.

Blood Platelets↗
PubMed1986

Phosphorylation of the 47 KDa protein in gamma-thrombin-stimulated human platelets does not activate phospholipase A2: evidence against lipocortin.

Intact human platelets were stimulated with alpha or gamma thrombin in the presence and absence of epinephrine and the ability of these agonists to stimulate aggregation, arachidonic acid release and protein phosphorylation was measured. Epinephrine alone had no effect on any of these events. Both alpha and gamma thrombin induced platelet aggregation which was potentiated in each case by epinephrine. Similarly, both thrombin species were able to induce the phosphorylation of platelet 20 KDa and 47KDa proteins. The gamma thrombin-induced phosphorylation was slightly enhanced by epinephrine. In contrast, only alpha thrombin was capable of inducing significant arachidonic acid release and the small release induced by gamma thrombin was reduced by epinephrine. These results show that the agonist-induced phosphorylation of the 47KDa protein by protein kinase C does not impart the ability to activate phospholipase A2 in human platelets, and questions the suggestion that the 47KDa protein is lipocortin.

Annexins↗
PubMed1985

The effects of gamma-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini.

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, gamma-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca2+ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.

Amylases↗
PubMed1982

Developmental loss of a smooth muscle muscarinic receptor population.

The radioligand [3H]quinuclidinyl benzilate has been used to examine the muscarinic receptor population of the chick expansor secundariorum muscle during its post-hatch development. At hatch the muscle bound 0.165 fmol of the ligand, but during the following two weeks binding decreased rapidly. Further reduction in ligand binding to the adult stage was observed, but at a much slower rate. The developmental loss of binding sites within this tissue corresponds to previously demonstrated reduction in acetylcholine sensitivity.

Aging↗
PubMed1981

Mepacrine inhibition of bradykinin-induced contractions of the rabbit ear vein.

Bradykinin induced concentration-dependent contractions in spiral strip preparations of the central vein of rabbit's ears. These contractions require the presence of Ca2+ in the extracellular medium. Mepacrine (2.1 to 42.3 mumol/l) significantly reduced these contractions. Since indomethacin (28 mumol/l) did not reduce the contractions produced by bradykinin, the effect of mepacrine is not explained by inhibition of prostaglandin synthesis. Procaine (10(-7) to 10(-4) mol/l) did not reduce the contractions produced by bradykinin and thus is does not seem that mepacrine acts through a non-specific, local anaesthetic effect. Mepacrine reduced the phosphatidate-mediated transfer of 45Ca from an aqueous phase to an organic phase. It has recently been proposed that phosphatidate may be the Ca2+ ionophore in smooth muscle. However, in the present experiments procaine was about as potent as mepacrine in inhibiting phosphatidate-mediated 45Ca transfer, and since it did not reduce the contractile responses these results do not support the concept that mepacrine inhibits contractions by reducing the Ca2+ influx via a phosphatidate ionophore.

Animals↗