Yigal H. Ehrlich

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

Abstract: The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 μM. The present study examined the effects of extracellular ATP in the concentration range of 0.1–1.0 mM on second‐messenger‐generating systems in cultured neural cells of the clones NG108‐15 and NIE‐115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin‐2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCI. Extracelluar ATP induced a concentration‐dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin‐2 signals. At the same concentration range (0.1–1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1‐stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15‐fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG 108–15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half‐maximal effects on Ca2+ uptake. Extracellular ATP at concentrations that can be produced in the synaptic cleft by repetitive stimulation but not during routine transmission can thus initiate a unique chain of events, which may play a role in the induction of long‐term adaptive changes in neuronal function.

ATP secretion and extracellular protein phosphorylation by CNS neurons in primary culture

The significant role of secreted ATP in the regulation of neuronal function and the activity of ecto-protein kinases which utilize extracellular ATP to phosphorylate proteins localized at the cell surface have been previously studied in peripheral neurons and in cloned neural cell lines. In the present study we have utilized neostriatal neurons differentiated in primary culture to demonstrate vesicular secretion of ATP and phosphorylation of proteins by extracellular ATP in neurons derived from the central nervous system (CNS). Neostriatal neurons from embryonic mice were maintained in a chemically defined medium for 15-18 days. Functional differentiation was determined by measuring evoked GABA-release. ATP-secretion was measured by luciferin-luciferase assays, and protein phosphorylation by adding gamma-32P-ATP to the extracellular medium. Depolarization by 50 mM KCl induced a Ca++-dependent ATP release, and stimulation by 100 microM veratridine resulted in secretion of ATP that could be blocked by tetrodotoxin. Phosphorylation of specific protein components with apparent molecular mass of 110 Kd, 80 Kd, 55 Kd, 30 Kd and 20 Kd was detected in striatal neurons incubated for 15 min with gamma-32P-ATP added to the medium, but not by labeling intracellular ATP pools with equivalent amounts of radioactivity presented as inorganic 32Pi. These results open for investigation the role of extracellular protein phosphorylation systems in processes underlying the responsiveness of CNS neurons to secreted ATP.

Regulation of Norepinephrine Uptake by Adenine Nucleotides and Divalent Cations: Role for Extracellular Protein Phosphorylation

Abstract: This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high‐affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5′‐adenylyl‐imidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+‐stimulated uptake 1 was blocked by App(NH)p; Mg2+‐stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca‐omission, and inhibition of the phosphorylation of one specific, 39,000‐dalton, Ca2+‐dependent, protein component in synaptosomes. This 39K protein was distinct from the α subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.

Interactions of the Alkyl-Ether-Phospholipid, Platelet Activating Factor (PAF) with Platelets, Neural Cells, and the Psychotropic Drugs Triazolobenzodiazepines

PAF-acether, a naturally occurring phospholipid, is a potent activator of various biological processes, including platelet aggregation. The mechanisms of action of PAF are largely unknown. We have found that the psychotropic triazolobenzodiazepine drugs, alprazolam and triazolam, potently (IC50 less than 1 microM) inhibit PAF-induced shape change, aggregation and secretion of human platelets. These effects are specific for PAF-activation, since the responses of human platelets to other agonists (ADP, thrombin, epinephrine, collagen, arachidonate and the Ca++ ionophore, A23187) are not inhibited by these triazolobenzodiazepines. The action of triazolobenzodiazepines on PAF-induced platelet function has clinical relevance, especially in diseases where enhanced platelet aggregability may lead to thrombosis and atherosclerosis. In addition, the ability of triazolobenzodiazepines to inhibit other PAF-mediated cellular-responses, such as anaphylactic shock or bronchoconstriction, suggests that these drugs may be useful in preventing several known pathophysiological effects of PAF. The specific antagonism of PAF action by psychotropic drugs also suggests that PAF or PAF-like phospholipids may play a role in neuronal function. This possibility was tested by examining the effects of PAF on neural cells of the clonal line NG108-15, grown in culture in a chemically defined, serum-free medium. Low concentrations of PAF (0.5-2.5 microM) induced neurite extension in NG108-15 cells, whereas higher concentrations (greater than 3 microM) were cytotoxic. Using NG108-15 cells preloaded with aequorin, it was found that PAF causes an increase in intracellular ionized calcium concentration, which is dependent on the presence of extracellular calcium. These results suggest that PAF-induced Ca++ uptake may play a role in neuronal development, and that circulating PAF may contribute to the neuronal degeneration caused by the exposure of neural tissues to blood in situations such as spinal cord injury, trauma, or stroke.

A Ca2+‐Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

Abstract: The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5‐hydroxytryptamine (5‐HT, serotonin). A protein kinase activity that copurifies with SBP (SBP‐kinase) was partially characterized and compared with calcium/calmodulin‐dependent protein kinase II (CAM‐PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5‐HT. SBP‐kinase and CAM‐PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+‐dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule‐associated proteins well and did not phosphorylate myosin, phosphorylase b., and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP‐kinase and CAM‐PK II were observed: (1) CAM enhanced CAM‐PK II activity, but had no effect on SBP‐kinase; (2) synapsin I was an excellent substrate for CAM‐PK II, but not for SBP‐kinase; (3) 5‐HT inhibited both the autophosphorylation of SBP‐kinase and the phosphorylation of SBP, but had no effect on CAM‐PK II. These data indicate that SBP‐kinase is different from CAM‐PK II. Phosphopeptide maps of SBP and SBP‐kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5‐HT within neurons.

Protein Phosphorylation in the Regulation and Adaptation of Receptor Function

Publisher Summary Phosphorylation and dephosphorylation of proteins play important and ubiquitous roles in the regulation of neural function. This evidence is based on two lines of investigation. The first is the demonstration that various neurotransmitters, neurohormones, and neurotropic factors, either directly or indirectly, regulate the phosphorylation of different proteins in neural tissue. The second line of investigation has demonstrated that various neuronal processes are regulated by phosphorylative activity. These include the metabolism and release of neurotransmitters, the induction and biosynthesis of neural-specific enzymes, axonal transport, chemically and electrically induced depolarization, receptor sensitivity, neuronal maturation and differentiation. Moreover, reports from several laboratories have implicated modifications in protein phosphorylation systems in processes whereby neurons respond with long-lasting alterations to persistent environmental, hormonal or pharmacological stimulations. In addition, several key cellular processes, such as DNA and RNA synthesis, metabolism of carbohydrates, and lipids, membrane permeability, mitochondrial function, cell division, and transitions in cell cycle have been shown to involve protein phosphorylation.

Activation of Adenylyl Cyclase by Preincubation of Rat Cerebral‐Cortical Membranes Under Phosphorylating Conditions: Role of ATP, GTP, and Divalent Cations

Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation‐dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral‐cortical membranes with adenosine‐5′‐0‐(3‐thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine‐5′‐0‐(3‐thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATPγS into a protein component with apparent Mr of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATPγS. Endogenous activity utilizing [γ‐32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane‐bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP‐preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.

Extracellular Protein Phosphorylation in Neuronal Responsiveness and Adaptation

The significant role of protein phosphorylation systems in the regulation and modulation of multiple neuronal functions has been extensively documented in numerous studies over the last three decades. Beginning with the demonstration by Heald (1957) that brief depolarization of respiring brain slices can cause a significant increase of phosphate incorporation into cerebral proteins, progress in this line of investigation has led to the conclusion that the cyclic process of phosphorylation/dephosphorylation of proteins represents a ubiquitous target for diverse agents which produce rapid and transient changes in neuronal activity (reviewed by Greengard, 1978; Rodnight, 1983). More recent studies, carried out with identified neurons of invertebrates, have begun to provide direct evidence for the role of specific phosphoproteins in certain well defined neuronal functions (reviewed by Nestler and Greengard, 1983). Thus, in the chain of events that occurs intracellularly subsequent to the activation of second-messenger generating systems by neurotransmitters, hormones, growth factors and trophic agents, phosphoproteins constitute a crucial link essential for the process of stimulus-response coupling (for most recent reviews see Nishizuka, 1986 and chapter by Greengard et al., in this volume). In addition, phosphorylative activity has been recognized as a site of molecular adaptation in neurons, since it was shown that modifications in the process of protein phosphorylation are induced by inputs which cause long-lasting alterations in brain function (reviewed by Ehrlich, 1979, 1984, see also chapters by Lovinger and Routtenberg and by Alkon and Naito in this volume).

GTP-preferring protein phosphorylation systems in brain membranes: possible role in adenylate cyclase regulation.

Abstract Preincubation of brain membranes with GTP under phosphorylating conditions resulted in activation of adenylate cyclase which withstood sedimentation and washing. Investigation into the possible mechanism(s) underlying this activation revealed that these membranes contain endogenous systems which prefer to utilize GTP, rather than ATP, in the phosphorylation of specific protein substrates with apparent M.W. of 54K and 33K. This activity is highly stimulated by Mn++ ions, inhibited by cyclic AMP and independent of Ca++. Triton-X-100 extracts of brain membranes, which contain the catalytic and regulatory subunits of adenylate cyclase, were found to be enriched in endogenous activity which phosphorylated the 54K protein with GTP, but not ATP. These findings provide a means for direct testing of the hypothesis that protein phosphorylation plays a role in adenylate cyclase regulation.

Chapter 17 Extracellular protein phosphorylation systems in the regulation of neuronal function

Publisher Summary This chapter presents evidence consistent with the suggestion that the modulation of certain neuronal functions by extracellular adenosine triphosphate (ATP) involves the activity of extracellular protein kinases, which phosphorylate proteins localized at the outer surface of the plasma membrane of neural cells. Several lines of investigation have provided evidence that extracellular ATP exerts potent effects on the activity of excitable cells, neurons and muscle. In addition to its role as a neurotransmitter, extracellular ATP can also serve as a modulator, regulating the activity of other neurohormones. One well-documented modulatory effect of extracellular ATP is the inhibition of evoked quantal acetylcholine secretion. Recent studies have demonstrated that when ATP is applied extracellularly to preganglionic nerves, it inhibits acetylcholine release. The cells of the neuroblastoma X glioma hybrid line, NG108-15, grown and differentiated in a chemically defined medium, are used to demonstrate the presence of an ecto-protein kinase and specific substrates for its activity at the surface of neural cells.