Modulation of amino acid neurotransmitter actions by other neurotransmitters: some examples.

Developments in the field of central neurotransmission indicate that amino acids serve as important and widespread transmitters throughout the central nervous system. There are increasing indications from recent experimental studies that several of the other central neurotransmitters may exert potent effects on central neurons by modulating the actions of amino acids. Noradrenaline and serotonin have received particular attention as potential modulators, and a wide variety of actions has been reported for them. Modulatory actions have been reported at both pre- and post-synaptic levels, including both short- and long-term effects and facilitation or inhibition of amino acid actions. Selectivity has been found both for specific receptor subtypes of the neuromodulator and for specific effects of amino acids. Examples of such selectivity are modification of actions of an amino acid with little effect on spontaneous activity or membrane properties of the target cell, or in comparison to the actions of other neurotransmitters, or even other selective amino acid analogs. Modulatory actions on amino acids have also been reported for several other neurotransmitters including acetylcholine and various peptides. Recent studies of angiotensin II demonstrate that when iontophoretically applied, it can potently and selectively block the depolarizing action of glutamate on locus coeruleus neurons. It is possible that physiological influences of these various transmitter substances are expressed through modification of amino acid actions, rather than through direct effects on central neurons.     

Thyrotropin releasing hormone stimulation of GABA-gated but not basal chloride ion influx in rat cerebellum

Potential interaction between gamma-amino butyric acid (GABA) and thyrotropin-releasing hormone (TRH) in eliciting a variety of central nervous system (CNS)-related biologic activities is well known; however, the mechanism underlying this interaction is not clearly defined. To gain further insight into this interaction, we examined the effects of TRH and two of its central nervous system selective analogs, DN 1417 and TA 0910, on basal and GABA-mediated chloride ion influx into rat cerebellar neurosynaptosomes. The results of these studies show that TRH may facilitate GABA action by augmenting chloride ion influx and hyperpolarization of cerebellar neurons.     

Enhanced neuronal K+ conductance: a possible common mechanism for sedative-hypnotic drug action

It is commonly thought that central nervous system depressant drugs exert their actions through enhancement of gamma-aminobutyrate (GABA)-mediated mechanisms. Recently, the cellular electrophysiological evidence from this laboratory and others suggests that both sedative hypnotics and general anaesthetics inhibit central neurons by increasing potassium conductance (GK). We have utilized the mammalian in vitro hippocampal and cerebellar slice preparations at 34-36 degrees C. Intracellular recordings from CA1, CA3, and cerebellar Purkinje cells were obtained. Low dose (sedative) concentrations of ethanol (less than or equal to 20 mM), two different benzodiazepines (midazolam and clonazepam in low nanomolar concentrations), and pentobarbital (10(-6) to 10(-4) M) were applied by pressure ejection or were bath perfused. All drugs caused a hyperpolarization with decreased spontaneous activity, and enhanced post spike afterhyperpolarizations (AHPs). These long-lasting AHPs are presumably due to enhanced calcium-mediated GK. Increased responsiveness to focally applied GABA was only seen at higher doses (ethanol, 100 mM; midazolam, 10(-7) M; pentobarbital, 10(-4) M). These data suggest that the above neurodepressant drugs, when applied at sedative doses to hippocampal pyramidal cells, enhance GK and not the actions of GABA.     

Autoreceptors do not regulate routinely neurotransmitter release: focus on adrenergic systems

The theory that neurotransmitter release is regulated locally at the individual terminals of neurons has achieved a rapid and seemingly secure status in our understanding of neuronal function both in the periphery and in the central nervous system. This concept of negative feedback control through the monitoring of the perineuronal concentration of previously released transmitter has been extended to a multiplicity of transmitters and utilized to explain the mechanisms of action of diverse classes of drugs, ranging from antihypertensives to antidepressants. It is my view that negative feedback by terminal and by somadendritic receptors cannot account for the existing body of experimental work. Analyses of the profiles of action of agonists and antagonists, and of the per pulse release of transmitter in the absence of drugs in a variety if peripheral organ systems, as well as in superfused brain slices, demonstrates the need for alternate interpretations of the available data. Evidence is provided that the actions of agonists to inhibit transmitter release and that of antagonists to enhance release occur at different cellular loci and that the purported unitary action of these two classes that is so central to the validity of presynaptic theory is unsupportable.     

GABA generates excitement

In the CNS, gamma-aminobutyric acid (GABA) acts as an inhibitory transmitter via ligand-gated GABA(A) receptor channels and G protein-coupled GABA(B) receptors. Both of these receptor types mediate inhibitory postsynaptic transmission throughout the nervous system. For GABA(A) receptors, this inhibitory action is associated with a hyperpolarization due to an increase in conductance to chloride ions. Previous studies show that GABA(A) receptor activation in neonatal neurons and spinal cord neurons can be excitatory. Two papers recently appeared that clearly demonstrate that GABA can have a depolarizing and excitatory action in mature cortical neurons. Here we discuss the evolving story on chloride ion homeostasis in CNS neurons and its role in the bipolar life of the GABA(A) receptor.     

Adrenergic stimulation of hepatocyte growth factor expression.

Hepatocyte growth factor (HGF), a potent mitogen, is released into plasma at increased levels following injury to certain tissues, including the liver. Early increases in plasma HGF are not due to a release from the injured liver, but rather from distal organs, particularly the lung. We have investigated the ability of norepinephrine (NE), which rises rapidly in plasma after liver resection, to trigger elevated production of HGF in MRC-5 human embryonic lung fibroblasts. Levels of HGF released to culture media and of HGF mRNA increased when cultures were exposed to NE, or to other adrenergic agonists. While stimulation of either beta- or alpha(1)-adrenergic receptors increased HGF expression, responses to NE appear to be mediated primarily via beta receptors. Since NE has already been shown to act as a comitogen with HGF, our findings suggest that adrenergic hormones may act both to induce production of HGF at distal sites, and to enhance the response to HGF at target tissues.     

A pro-survival effect of polyamine depletion on norepinephrine-mediated apoptosis in cardiac cells: role of signaling enzymes

Recent studies report that the primary transmitter of sympathetic nervous system norepinephrine (NE), which is actively produced in failing human heart, is able to induce apoptosis of rat cardiomyocytes. Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, therefore representing a potential therapeutic target. The natural occurring polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. Thus, we have studied the involvement of polyamines in the apoptosis of cardiac cells induced by the treatment with NE. The results indicate that NE caused an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of apoptotic cell death. This effect was prevented in the presence of α-difluoromethylornithine, an irreversible inhibitor of ODC. Moreover, the study of some key signal transduction pathways revealed an involvement of AMP-activated protein kinase, AKT and p38 mitogen-activated protein kinases, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact, polyamine-depleted cells showed an altered activation pattern of these kinases that may contrast apoptosis and appeared to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. In conclusion, these results indicate that in cardiac cells polyamines are involved in the execution of the death program activated by NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.     

Ephaptic feedback in identified synapses of terrestrial snails

A hypothesis for the existence of the intrasynaptic ephaptic feedback (EFB) in the invertebrate central nervous sytem was tested. Excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) evoked by the activation of the recently described monosynaptic connection between the identified snail neurons were recorded intracellularly. In case of the EFB presence, the postsynaptic tetanization with hyperpolarization pulses could activate presynaptic Ca2+ channels and enhance the EPSP amplitude, whereas a steady postsynaptic hyperpolarization should induce a "supralinear" increase in EPSC amplitudes as it has been found in the rat hippocampus. In the first series of the experiments, 10 trains of hyperpolarizing pulses (40-50 mV, 1 Hz, pulse duration 0.5 s, train duration 45 s) were delivered postsynaptically. No significant changes in EPSP amplitudes were found. In the second series of the experiments, the EPSC amplitudes were measured during varying postsynaptic hyperpolarization. At the membrane potential 100 mV, the EPSP amplitude was significantly higher than theoretically predicted from the classical linear dependence. Such a "supralinear" effect of postsynaptic depolarization can be explained by the presence of the EFB. This finding is the first evidence for the EFB existence in the invertebrate central nervous system.     

Possible role of cyclic AMP and dopamine in morphine tolerance and physical dependence.

In chronically morphinized rats undergoing naloxone induced withdrawal the cerebellar Cyclic 3′, 5′ adenosine monophosphate (Cyclic AMP) was significantly higher than the controls. The cerebellar dopamine (DA) and norepinephrine (NE) were decreased, elevated or unchanged depending on the duration of morphine treatment. The corpus striatal DA levels during withdrawal were markedly elevated and the striatal cyclic AMP levels were unchanged. The NE levels in the striatal tissue were either elevated or unchanged depending upon the duration of morphine administration. In sharp contrast to the chronically morphinized rats undergoing naloxone induced withdrawal, the rats made morphine dependent over a period of eight weeks showed quite moderate changes in the striatal and cerebellar cyclic AMP and DA levels. Thus alterations in the DA and the cyclic AMP levels in the central nervous system (CNS) may play an important role in the naloxone induced stereotyped morphine withdrawal behavior.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

Kainic acid and synaptic transmission in the stellate ganglion of the squid.

Kainate, a conformational analogue of glutamate, blocks synaptic transmission across the giant synapse of the squid. In the presence of blocking doses of kainate, impulses continue to propagate into the nerve terminal, but action potentials are slightly reduced in size and the subsequent hyperpolarization is greatly diminished. Kainate depolarizes the postsynaptic axon. Since the depolarizing action of kainate is confined to the postsynaptic membrane, it appears that kainate can combine with the receptors which are normally activated by the transmitter. This results in a diminished effect of the transmitter released by a presynaptic nerve impulse.     

Peripheral Actions of the SCPs in Aplysia and Other Gastropod Molluscs

The SCPs are a family of neuropeptides found in many gastropodspecies. Two SCPs with similar sequences have been characterizedin Aplysia. These peptides are potent modulators of centraland peripheral synapses. They also enhance ongoing contractileactivity in spontaneously active tissues such as heart and gut.Their distribution in central ganglia suggests that their predominantrole is in the regulation of feeding behavior. There is goodevidence that the identified SCP-containing neurons, B1 andB2, provide the major central regulation of gut motility duringfeeding through the release of the SCPs from their terminalsin gut. The SCPs have also been localized to motor neurons thatinnervate buccal muscles which generate biting and swallowingmovements. In many of these neurons, the SCPs have been shownto coexist with conventional transmitters such as ACh, or otherpeptides such as FMRFamide. The SCPs appear to be released alongwith conventional transmitters from these neurons to modulatethe effectiveness of the conventional transmitter. In all cases,the SCPs cause an enhancement of the amplitude of contractionsproduced by motor neuron stimulation. The precise mechanismsunderlying this effect vary from muscle to muscle. All of theeffects of the SCPs are mediated by increased cAMP levels intarget tissue. At many sites of action, serotonin produces actionsthat are qualitatively similar to those of the SCPs. This islikely to involve a convergence at the level of the adenylylcyclase. In addition to these peripheral effects, the SCPs alsohave multiple central effects on feeding and other behaviorsin gastropods.     

Na+/K(+)-ATPase as an effector of synaptic transmission.

Evidence is presented in support of the hypothesis that transmitter monoamines can exert their post-synaptic effects by stimulation or inhibition of Na+/K(+)-ATPase in neuronal or glial cell plasma membranes. Stimulation of electrogenic sodium pumping, causing a hyperpolarization with an increase in membrane resistance, could account for the depression of neuronal spontaneous firing and the signal/noise enhancing actions of these amines. Conversely, inhibition of an electrogenic sodium pump in neuronal plasma membranes would lead to depolarization and enhanced excitability.     

Electrophysiological actions of neurotensin in rat cerebellum

The electrophysiological actions of neurotensin (NT) and its analog d-Arg⁹NT were studied in rat cerebellar Purkinje neurons. NT applied by pressure ejection was a potent depressant of Purkinje (P) neuron firing. In contrast, iontophoretically applied NT was a weak depressant. Pressure-ejected d-Arg⁹NT, which is largely inactive in peripheral systems, had little effect on P neurons. The depressant effects of pressure-ejected NT were blocked by intraperitoneally administered haloperidol, iontophoretically applied magnesium or 6-OHDA pretreatment. After such treatments, locally applied NT evoked only excitations.The results of this study suggest that NT, when applied by pressure ejection, produces two effects on the Purkinje neuron. The potent inhibitory effects of locally applied NT appear to result from release of the inhibitory transmitter, norepinephrine from locus coeruleus-derived afferents. We postulate that the excitations, which appear when postsynaptic effects of norepinephrine are antagonized or release is reduced, may be the direct result of NT action at the postsynaptic P neuron membrane.     

Effects of acetylcholine on the coronary artery

Acetylcholine acts on the different components of the coronary arterial wall by 1) initiating endothelium-dependent relaxation of the smooth muscle cells; 2) inhibiting the exocytotic release of norepinephrine (NE), which could result in either vasodilator or vasoconstrictor effects depending on whether the main action of NE is alpha- or beta-adrenergic, respectively; and 3) activating the contractile process of the smooth muscle cells. These different effects of the cholinergic transmitter are muscarinic in nature. Their relative importance varies among species, or when acetylcholine is given exogenously rather than released from cholinergic nerves.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

On the origin of skewed distributions of spontaneous synaptic potentials in autonomic ganglia.

The histograms of spontaneous synaptic potentials at synapses in autonomic ganglia are described by distributions consisting of mixtures of Gaussians, rather than by single Gaussian distributions. The possible origin of these mixed distributions is investigated, using Monte-Carlo simulations of the action of spontaneously released units of transmitter. A single unit of acetylcholine of fixed size, released from an active zone with receptor patches both beneath and adjacent to the zone, does not give rise to the observed histograms. But if the unit is of variable size, consisting of integer multiples of smaller units, and release is from an active zone onto either the receptor patch beneath, or in addition onto adjacent patches, then the histogram is well described by a mixture of Gaussians. However, this explanation is unlikely to be correct as present evidence suggests that in most cases the released unit of transmitter saturates the postsynaptic receptor patch beneath the active zone. The final case considered is where a unit of transmitter is spontaneously released from an active zone, simultaneously with a unit in an adjacent zone less than one micron away. The histogram of potentials then conforms to those observed even when there are differences in the sizes of the receptor patches. It is suggested that this kind of release could provide an explanation for distributions of spontaneous potentials that are mixtures of Gaussians.     

ATP as a presynaptic modulator

There is considerable evidence that ATP acts as a fast transmitter or co-transmitter in autonomic and sensory nerves mostly through activation of ionotropic P2X receptors but also through metabotropic P2Y receptors. By analogy, the observations that ATP is released from stimulated central nervous system (CNS) nerve terminals and that responses to exogenously added ATP can be recorded in central neurons, lead to the proposal that ATP might also be a fast transmitter in the CNS. However, in spite of the robust expression of P2 receptor mRNA and binding to P2 receptors in the CNS, the demonstration of central purinergic transmission has mostly remained elusive. We now review evidence to suggest that ATP may also act presynaptically rather than solely postsynaptically in the nervous system.     

Effects of aging at the adrenergic cardiac neuroeffector junction

Adrenergic neural degeneration was seen to increase with age. This is thought to contribute to the decreased cardiac content of the transmitter. Pharmacologically, it was found with the use of tyramine that virtually all of the norepinephrine (NE) pool is available for release, and that there is no difference in the amount of NE released in relation to age. Cardiac responsiveness to adrenergic agonists decreases with age. Our results suggest that this is caused in great measure by increased activity of the prejunctional, neuronal uptake mechanism in the older animal.     

Diazepam potentiation of purinergic depression of central neurons.

Intravenously or iontophoretically applied diazepam potentiated the depressant action of iontophoretically applied 5'-AMP on the spontaneous firing of rat cerebral cortical neurons. This potentiation of purinergic depression may be a result of the previously reported inhibition by diazepam of uptake of adenosine into brain tissues.