Neuropeptide Y mobilizes intracellular Ca2+ and increases inositol phosphate production in human erythroleukemia cells

The intracellular concentration of free Ca2+ was monitored by measuring the fluorescence of fura-2 loaded Human Erythroleukemia Cells. Neuropeptide Y (NPY) increased intracellular Ca2+ in a dose-dependent manner and the 50% effective concentration was 2 nM. Chelation of extracellular Ca2+ by EGTA did not reduce the NPY-mediated increase in cytoplasmic Ca2+, indicating that the increase in fluorescence was due to the release of intracellular Ca2+. A second dose of NPY, after intracellular Ca2+ had returned to basal levels, failed to elicit a response, indicating that the NPY receptor had undergone desensitization. In similar experiments, NPY increased the formation of inositol phosphates, suggesting that the mobilization of Ca2+ from intracellular stores in HEL cells was secondary to the generation of inositol phosphates and stimulation of phospholipase C.     

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.     

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.     

GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+

The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C.     

Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.     

Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets

The ability of exogenous sn-1,2-diacylglycerols and analogs to function as bioregulators of protein kinase C in human platelets was investigated. The activation of protein kinase C in platelets is indicated by specific phosphorylation of a 40,000-dalton protein. Dihexanoylglycerol, dioctanoylglycerol (diC8), didecanoylglycerol, and sn-1-oleoyl-2-acetylglycerol were active in stimulating 40,000-dalton protein phosphorylation. Only a trace of phosphorylation was elicited by dibutyrylglycerol. Phosphorylation was not induced by analogs of diC8 in which an -H, -SH, or -Cl group replaced the free -OH, nor by monoacylglycerols or long chain diacylglycerols. Maximum phosphorylation was induced by dihexanoylglycerol, diC8, and didecanoylglycerol at concentrations from 5 to 20 microM and between 5 and 30 S after exposure of platelets to these diacylglycerols. Under conditions of maximal phosphorylation of the 40,000-dalton protein, these diacylglycerols did not induce phosphatidylinositol turnover, or platelet aggregation, or stimulate release of ATP or serotonin. A small degree of aggregation was evident with platelets isolated in the absence of prostacyclin, and release of serotonin was observed when 1 mM Ca2+ or submaximal concentrations of ionophore A23187 were included. These results are consistent with a model in which platelet activation requires the simultaneous formation of two intracellular signals, diacylglycerols and Ca2+. These diacylglycerols and diacylglycerol analogs provide useful tools to investigate the function of diacylglycerols as bioregulators in intact cells.     

Collagen stimulates [3H]inositol trisphosphate formation in indomethacin-treated human platelets.

The present study investigates the pathway of metabolism of inositol phospholipids in human platelets exposed to collagen. Platelet activation by collagen was preceded by a lag phase usually lasting 10-20 s. Formation of [3H]inositol trisphosphate (IP3) was not observed during this period, but occurred in parallel with the onset of aggregation, release of ATP and phosphorylation of a 20 000 Da and a 40 000 Da protein. Indomethacin treatment partially inhibited all of these responses. Aggregation and ATP release, but not IP3 formation, were further inhibited in indomethacin-treated platelets loaded with the fluorescent Ca2+ indicator, quin2. Under these conditions there was no detectable mobilization of Ca2+. These results demonstrate that activation of platelets by collagen is associated with rapid hydrolysis of polyphosphoinositides by phospholipase C, thereby producing IP3. This observation is discussed in relation to IP3 as a possible Ca2+-mobilizing agent.     

Dual mechanisms of platelet hormone receptor desensitization. Differential importance between agonists of protein kinase C-dependent and -independent pathways

Several different agonists, among them alpha-thrombin, platelet-activating factor, vasopressin, thromboxane A2, and endoperoxides, activate platelets to aggregate and secrete granular contents. Each of these agents is thought to act by inducing the turnover of inositol phospholipids and generating the second messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. However, within minutes, the action of these agonists desensitizes. We have studied the characteristics of this desensitization process for the agonists mentioned above in an attempt to clarify the mechanisms involved. Our results show that two different pathways of desensitization exist, one that is mediated by protein kinase C and another that is independent of this enzyme. In addition, the contribution of these pathways to desensitization differs for the agonists studied. Our data suggest that partial agonists and strong agonists differ in the rate at which the primary response is desensitized rather than in their ability to couple to phospholipase C.     

Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells

C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.     

Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.