Effect of ICV pertussis toxin and N-ethylmaleimide on the levels of substance P and serotonin in brain and spinal cord of the rat.

The effects of single intracerebroventricular (icv) injections of either 0.5 microgram pertussis toxin or 5 micrograms N-ethylmaleimide (NEM) on the levels of immunoreactive substance P (ir-SP) and serotonin (5-HT) in the brain and spinal cord of rats have been assessed. At two and six days after pertussis toxin injection, the levels of ir-SP appeared significantly diminished in the spinal cord (about 34%). This reduction was even greater at two days after NEM injection (43%). These two agents did not alter the ir-SP of the midbrain and thalamus, whereas NEM increased the neuropeptide content in the pons-medulla. On the other hand, the thalamic content of serotonin was reduced two days after pertussis toxin (32%) or NEM (20%) injection. The indoleamine levels of the spinal cord were reduced by these treatments (20%), while in the midbrain a slight decrease could be observed. These findings suggest that pertussis toxin and NEM produce these effects by acting upon a common neural substrate.     

Cholera toxin and pertussis toxin on opioid- and alpha 2-mediated supraspinal analgesia in mice.

Cholera toxin, an agent that impairs the function of Gs transducer proteins, was injected (0.5 microgram/mouse, icv) and the antinociceptive activity of opioids and clonidine was studied 24h later in the tail-flick test. In these animals, an enhancement of the analgesic potency of morphine, beta-endorphin and clonidine could be observed. Cholera toxin did not modify the antinociception evoked by the enkephalin derivatives DAGO and DADLE. Pertussis toxin that catalyses the ADP ribosylation of alpha subunits of Gi/Go regulatory proteins was given icv (0.5 microgram/mouse). This treatment reduced the analgesic effect of opioids and clonidine. However, while the analgesia elicited by DAGO, DADLE and clonidine was greatly decreased, the effect of morphine and beta-endorphin was reduced to a moderate extent. It is concluded that Gi/Go regulatory proteins functionally coupled to opioid and alpha 2 receptors are implicated in the efficacy displayed by opioids and clonidine to produce supraspinal analgesia. Moreover, these two receptors are susceptible to regulation by a process that might involve a Gs protein.     

Multiple signal transduction pathways mediated by 5-HT receptors

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.     

Effects of neonatal maternal separation on neurochemical and sensory response to colonic distension in a rat model of irritable bowel syndrome

Early life stress has been implicated as a risk factor for irritable bowel syndrome (IBS). We studied the effect of neonatal maternal separation on the visceromotor response and the expression of c-fos, 5-HT, and its receptors/transporters along the brain-gut axis in an animal model of IBS. Male neonatal Sprague-Dawley rats were randomly assigned to a 3-h daily maternal separation (MS) or nonhandling (NH) on postnatal days 2-21. Colorectal balloon distention (CRD) was performed for assessment of abdominal withdrawal reflex as a surrogate marker of visceral pain. Tissues from dorsal raphe nucleus in midbrain, lumbar-sacral cord, and distal colon were harvested for semiquantitative analysis of c-fos and 5-HT. The expression of 5-HT expression, 5-HT3 receptors, and 5-HT transporter were analyzed by RT-PCR. Pain threshold was significantly lower in MS than NH rats. The abdominal withdrawal reflex score in response to CRD in MS rats was significantly higher with distension pressures of 40, 60, and 80 mmHg. In MS rats, the number of c-fos-like immunoreactive nuclei at dorsal horn of lumbar-sacral spinal cord increased significantly after CRD. 5-HT content in the spinal cord of MS rats was significant higher. In the colon, both 5-HT-positive cell number and 5-HT content were comparable between MS and NH groups before CRD. Post-CRD only MS rats had significant increase in 5-HT content. Protein and mRNA expression levels of 5-HT3 receptors and 5-HT transporter were similar in MS and NH rats. Neonatal maternal separation stress predisposes rats to exaggerated neurochemical responses and visceral hyperalgesia in colon mimicking IBS.     

100Hz电针刺受时大鼠脑和脊髓k受体结构特性的改变

西方采用放射配体结合实验研究了１００ＨＺ电针耐受发生发展过程中大鼠脑和脊髓Ｋ受体结构特性的变化。大鼠每天给予１００ＨＺ电针１次,连续７ｄ。分别在电针的第１、３、５、７天取不同脑区进行观察。     

Monoamine and amino acid content in brain regions of Brattleboro rats

Monoamine and amino acid content were measured in brain regions from 12 week old male, homozygous Brattleboro (DI,n=12) and Long-Evans control (LE,n=12) rats. Norepinephrine (NE) content was significantly elevated (16–25%) in the spinal cord, pons-medulla and anterior hypothalamus of DI rats when compared to LE controls. NE content of the neurointermediate lobe of pituitary in DI rats was almost twice that of LE controls. Serotonin content was also significantly elevated in the spinal cord, pons-medulla, anterior hypothalamus and forebrain of DI rats relative to the LE controls. Taurine content in DI rats was increased (31–42%) above that of LE rats in the anterior hypothalamus, striatum and forebrain. Glutamine content was also greater in DI rats than LE in the spinal cord, pons-medulla, anterior hypothalamus, striatum, hippocampus and forebrain. The changes in monoamine and amino acid content were discussed in relation to the cardiovascular and osmoregulatory deficits that are present in DI rats due to arginine vasopressin (AVP) deficiency. The possible role of AVP in modulating NE turnover was also discussed. The increase in brain TAU content in DI rats may be a physiological response to hypernatremia.     

Functional Reconstitution of Purified Gi and Go with μ-Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N-Ethylmaleimide

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.     

Morphine tolerance in arthritic rats and serotonergic system

To understand whether chronic inflammation alters the development of morphine tolerance, the tail-flick test was used to evaluate the analgesic effect of morphine (75 mg tablet, s.c.) in the arthritic rats at the day 9-12 after the inoculation with Freund's adjuvant. Spinal cord monoamines and amino acid neurotransmitters were concomitantly measured. Chronic inflammation attenuated the antinociceptive effect of morphine as tolerance developed faster in the arthritic rats compared to the vehicle-treated controls. In addition, ratio of 5-hydroxyindole-3-acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) increased in the lumbar spinal cord of arthritic rats without any change in the concentrations of norepinephrine, glutamate, aspartate or GABA. Interestingly, increased serotonin turnover in the spinal cord was observed in both control and arthritic rats 24 hours after morphine treatment. Overall, the results suggest a significant role of serotonin up-regulation in the spinal cord during chronic pain and the development of morphine tolerance.     

Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.     

Nifedipine-sensitive vascular reactivity of femoral arteries in WKY: the effects of pertussis toxin pretreatment and endothelium removal

Maintenance of norepinephrine (NE)-induced contraction is dependent on Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCC), which is opposed by nitric oxide. Adrenergic receptors are coupled with different G proteins, including inhibitory G proteins (Gi) that can be inactivated by pertussis toxin (PTX). Our study was aimed to investigate the effects of endothelium removal, PTX pretreatment and acute VDCC blockade by nifedipine on the contractions of femoral arteries stimulated by norepinephrine. We used 12-week-old male WKY, half of the rats being injected with PTX (10 microg/kg i.v., 48 h before the experiment), which considerably reduced their blood pressure (BP). Contractions of isolated arteries were measured using Mulvany-Halpern myograph. NE dose-response curves determined in femoral arteries from PTX-treated WKY rats were shifted to the right compared to those from control WKY. On the contrary, removal of endothelium augmented NE dose-response curves shifting them to the left. Acute VDCC blockade by nifedipine (10(-7) M) abolished all differences in NE dose-response curves which were dependent on the presence of either intact endothelium or functional Gi proteins because all NE dose-response curves were identical to the curve seen in vessels with intact endothelium from PTX-treated animals. We can conclude that BP reduction after PTX injection is accompanied by the attenuation of NE-induced contraction of femoral arteries irrespective of endothelium presence. Moreover, our data indicate that both vasodilator action of endothelium and Gi-dependent vasoconstrictor effect of norepinephrine operate via the control of Ca(2+) influx through VDCC.     

Brain somatostatin receptor-G protein interaction. G alpha C-terminal antibodies demonstrate coupling of the soluble receptor with Gi(1-3) but not with Go.

Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of low Km GTPase activity in mouse CNS: Effect of chronic morphine

The low Km GTPase displayed an apparent Km value of 0.2–0.4 μM in P₂ fractions from whole mouse brain. The activity of this enzyme ranged from 102 (pmols of GTP hydrolysed per μg of protein per min) in the striatum to 39 in the pons-medulla oblongata. Intermediate values were found in other structures, 74-62 in thalamus, hypothalamus, periaqueductal gray matter (PAG), rest of mesencephalon, cortex and spinal cord. The Km also varied throughout the mouse CNS: the spinal cord, striatum and PAG exhibited Km values (0.308-0.271 μM) higher than cortex, thalamus, pons-medulla, hypothalamus and remaining mesencephalon (0.239-0.193 μM). Chronic morphine (3 days) decreased the low Km GTPase activity of PAG (42), whereas it increased the one of thalamus (99). After chronic exposure to the opioid the Km values of the enzyme in striatum (0.193), PAG (0.192) and spinal cord (0.201) diminished, and the ones of hypothalamus (0.357) and rest of mesencephalon (0.287) augmented. The herein reported diversity of low GTPase activity might be due to the presence of different ratios of Gα types/subtypes in the neural structures studied. As a result of chronic morphine the ratio and/or the functionality of G proteins would be altered in particular areas of mouse CNS.     

Modification of Brain Guanine Nucleotide-Binding Regulatory Proteins by Tryptamine-4,5-Dione, a Neurotoxic Derivative of Serotonin

We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.     

Levels of norepinephrine and 5-hydroxytryptamine in the spinal cord of the dog following ischemia and recirculation

Forty minutes of abdominal aorta ligature (partial ischemia in the lumbosacral segments of the spinal cord) did not change significantly levels of norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in the spinal cord of the dog in comparison with sham-operated controls. After a 40-minute interval of recirculation NE levels dropped nonsignificantly below levels of the control group and 5-HT significantly in lumbar segments. It thus appears that even though in the stage of recirculation in the spinal cord energy metabolism is restored, the functional deficit of monoamines may persist.     

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.     

The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling

Using a universal signaling assay employing G-protein chimeras comprising the C-terminal five amino acids of Gi1/2, Gi3, Go, and Gz fused to Gq, the calcium mobilizing G-protein, we explored the role of the C-terminus of Gi family G-proteins as a determinant for 5-HT(1A) receptor functional coupling. Co-expression of the 5-HT(1A) receptor with each of the Gq/Gi family chimeras resulted in a concentration-dependent increase in calcium upon addition of 5-HT, although the coupling efficiency differed dramatically. Gq/Gi3 resulted in the most efficient coupling based on both potency and relative maximum response to 5-HT. Gq/Go also produced efficient coupling in terms of relative 5-HT efficacy (76% of the Gq/Gi3 maximum response), although 5-HT exhibited 4-fold lower agonist potency, and Gq/Gz and Gq/Gi1/2 conferred poor functional coupling. Agonist potencies and relative efficacies determined for a number of 5-HT(1A) receptor agonists using Gq/Gi3 coupling were significantly weaker than those described previously for coupling through the native G-protein. These results indicate the C-terminus of Gi3 as an important determinant for coupling to the 5-HT(1A) receptor, while the reduced functional agonist activities suggest additional motifs participate in receptor/G-protein coupling.     

Immediate and neurotoxic effects of HIV protein gp120 act through CXCR4 receptor

Primary rat hippocampal neurones show pronounced elevations of intracellular calcium within minutes of exposure to the HIV coat protein gp120. Culture of hippocampal neurones with gp120 causes significant neurotoxicity. We find that the peptide VSLSYRCPCRFF, a competitive inhibitor of the CXCR4 chemokine receptor, markedly inhibits toxicity and eliminates the acute calcium elevation. CXCR4 receptors are thought to signal to the Gi/Go family of trimeric GTP binding proteins. Pretreatment of hippocampal neurones with pertussis toxin to inactivate Gi/Go proteins markedly reduced gp120 neurotoxicity. These results indicate that both short and long term effects of gp120 are the result of activation of the CXCR4 receptor.     

Biochemical discrimination of the predominant pertussis toxin substrate of rabbit neutrophils from brain Gi and Go: isoelectric focusing improves resolution of pertussis toxin substrates

In the present study, the predominant pertussis toxin substrate in rabbit neutrophils, Gn, was biochemically compared to Gi and Go purified from brain, after being [32P]ADP-ribosylated by activated pertussis toxin and [32P]NAD. On SDS-polyacrylamide gels, a poorly resolved doublet from neutrophil membranes was observed; the upper band, corresponding to approximately equal to 25% labeling, comigrated with Gi-alpha and the predominant lower band, Gn, migrated intermediately between Gi-alpha and Go-alpha. Peptide maps generated by limited-digestion of the labeled Gn, Gi and Go with S. aureus V8 protease were slightly, but definitively and reproducibly different. Isoelectric focusing clearly distinguished Gn from the other two pertussis toxin substrates. The pI value of Gn, 5.60, was distinctly different from those of Gi, 5.75 and 5.80. Although the pI values for Go and Gn were similar (5.60), the patterns of the two proteins were qualitatively different, with Go being resolved into an equal doublet (pI = 5.55 and 5.60) while Gn appeared predominantly as a single band. Thus, Gn is biochemically distinguishable from Gi and Go of brain and these structural differences are most clearly evident following isoelectric focusing.     

THE EFFECT OF AMINO-OXYACETIC ACID ON BRAIN CATECHOLAMINES

Abstract— —Administration of amino-oxyacetic acid (AOAA) to rats induced a pronounced decrease of midbrain norepinephrine (NE) and adrenal epinephrine (E) after 30 min, at which time the GABA level of midbrain had increased to 117 per cent of the initial value. The concentrations of NE in the pons-medulla and of dopamine (DA) in the cerebral hemispheres were not changed.
Further increases in brain GABA were accompanied by a rise of NE in midbrain and pons-medulla beginning 1 hr after AOAA administration. A rise of cerebral DA level was observed only after 4 hr. Six hours after AOAA administration the levels of both NE and DA in brain were reduced.
From the results of these and other studies, where administration of small amounts of GABA were shown to affect brain NE and serotonin levels, it is suggested that monoamines may be involved in the physiological action of GABA in the brain.