Masato Ando

Alteration of Transglutaminase Activity in Rat and Human Spinal Cord after Neuronal Degeneration

We measured the activity of transglutaminase (TG), a Ca2+-dependent enzyme and a biochemical marker of cell degeneration, in the adult rat spinal cord after unilateral occlusion of a branch of the dorsal spinal artery, and compared it to the enzyme activity in the tissue on the contralateral side without ischemic damage. The affected half of the spinal cord showed a significant rise in intrinsic (endogenous) TG activity one day after ischemic insult while no apparent morphological changes were observed in the tissue. However, the enzymic activity on the affected side gradually decreased to reach the level in the non-affected tissue, accompanying severe degeneration of neuronal cells at 7 days after the surgery, then it declined to nearly half the level in the intact tissue 30 days after the operation. We also determined the TG activity in transverse sections of the human spinal cord obtained at autopsy from 5 amyotrophic lateral sclerosis (ALS) and 9 non-ALS patients. TG activity in thoracic and lumbar cords was markedly low in ALS patients not only in ventral and lateral regions but also in the dorsal portion. These findings imply that the reduced TG activity in the ALS spinal cord is one of the characteristic features of the disease reflecting exhaustion of the enzyme in the tissue resulting from degeneration of the spinal neurons through cross-linkage of soluble intraneuronal cytoplasmic proteins.

Rapid and transient alterations in transglutaminase activity in rat superior cervical ganglia following denervation or axotomy

The activity of transglutaminase (TG), a Ca(2+)-dependent enzyme contributing to cross-linkage formation of intracellular polypeptide chains decreased rapidly to ca. 25% of control level in superior cervical ganglia (SCG) within 0.5 h following denervation. The reduced level was maintained for at least 24 h. By contrast, following axotomy, ganglionic TG activity increased by ca. 50% within 1 h, maintained the increase to 4 h, and returned to control level by 24 h. When SCG were transferred to aerobic in vitro incubation conditions 3 h following denervation, the addition of the protein kinase C (PKC) inhibitor, trifluoperazine (TFP, 10 micrograms/ml), to the medium partially reversed the denervation-induced reduction in ganglionic TG activity. Addition of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA, 1 microM), had no effect on the TG activity. These findings suggest that the pathway resulting in the rapid, denervation-induced inhibition of TG activity may involve the transsynaptic activation of PKC. When SCG were placed in vitro 3 h following axotomy, addition of nerve growth factor (NGF, 0.25 micrograms/ml) to the medium reversed approximately one-half of the axotomy-induced increase in TG activity. Thus, following axotomy, the reduction in delivery to the SCG of NGF, which can be transported retrogradely within the axon and is indispensable for morphological and functional survival of sympathetic neurons, may trigger the transient, axotomy-induced TG activation in the SCG.

Selective alterations in transglutaminase activity of rat superior cervical ganglia in response to neurotransmitters, high potassium and sialic acid-containing compounds

We examined the in vitro effects of neurotransmitters, high KCl as well as sialic acid-containing compounds (GM1; SC) on transglutaminase (TG) activity in isolated superior cervical ganglia (SCG) one week after denervation or axotomy. Following denervation, TG activity in SCG decreased to 83% of the unoperated control value, whereas that of axotomized ganglia was 28% of control. Thus, TG activity was relatively unaffected when sympathetic ganglionic neurons were preserved, but was markedly reduced under conditions where neurons were degenerating. Addition of ACh (0.1 mM) to the medium during aerobic incubation stimulated TG activity more than 3-fold in denervated ganglia but had no effect on TG activity in axotomized ganglia. Similarly, the NE (0.05 mM)-induced decrease of TG activity observed in intact SCG was also seen following denervation (-49%) but not following axotomy. In denervated SCG, the stimulatory effects of ACh were virtually abolished by co-addition of the cholinergic antagonists, atropine or hexamethonium, while the suppressant effects of NE were blocked by the adrenergic antagonists, propranolol, prazosin or yohimbine. These results imply that transmitter-induced rapid changes in TG activity occur predominantly in ganglionic neurons. When the ganglia were depolarized by high KCl (50 mM), a significant increase in TG activity in each intact, denervated and axotomized SCG was seen with qualitatively similar manner, suggesting that high KCl-induced depolarization affects both neuronal and glial components in the SCG. The marked increase in ganglionic TG activity in response to GM1 (5 nM) and synthetic SC (0.02 mM) were lost in denervated SCG but only partially reduced in axotomized SCG.(ABSTRACT TRUNCATED AT 250 WORDS)

The intercellular communication via nitric oxide and its regulation in coupling of cyclic GMP synthesis upon stimulation of muscarinic cholinergic receptors in rat superior cervical sympathetic ganglia

Cyclic GMP (cGMP) production in rat superior cervical sympathetic ganglia (SCG) was markedly increased (ca. 7-9-fold) by the addition of either acetylcholine (ACh; 0.1 mM) or a muscarinic agonist, carbachol (Carb; 0.1 mM), in the presence of an inhibitor (3-isobutyl-1-methylxanthine) for cGMP hydrolytic enzyme during in vitro aerobic incubation at 37 degrees C for 5 min. The ACh-induced accumulation of cGMP in SCG was effectively blocked (-73%) by the further addition of atropine (10 microM), a muscarinic antagonist, whereas a nicotinic blocker, hexamethonium (10 microM) partially antagonized (-41%) this ACh stimulation. The inhibitory effect of hexamethonium on ACh-evoked ganglionic cGMP production was effectively augmented (-83%) by addition of NG-monomethyl-L-arginine (L-NMMA, 50 microM), a compound that inhibits nitric oxide (NO) synthesis from L-arginine. Comparable inhibition of cGMP formation was observed following application of L-NMMA to the SCG upon stimulation of Carb. In contrast, L-NMMA had no effect on the decreased level of ACh-evoked cGMP production caused by the muscarinic antagonist. The Carb-induced elevation of ganglionic cGMP synthesis was significantly reduced within 1 min of incubation in the medium containing hemoglobin (Hb; 20 microM), an agent that scavenges only the extracellular fraction of NO. Thereafter, the tissue cGMP formation attenuated to the control level by subsequent incubation for several minutes. Addition of protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) to the medium significantly decreased Carb-evoked cGMP synthesis (-61%) in SCG, whereas superoxide dismutase (SOD; 30 U/ml) only slightly suppressed the Carb stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation by acetylcholine and inhibition by norepinephrine of transglutaminase activity in superior cervical ganglia excised from adult rats

Very rapid changes in activity of transglutaminase (TG), a calcium-dependent enzyme contributing to cross-linkage formation of intracellular polypeptide chains, were observed in vitro in rat superior cervical ganglion (SCG) and nodose ganglion (NG) following application of cholinergic or adrenergic agonists and antagonists. In SCG, a tissue rich in synapses, the depolarizing agent acetylcholine (ACh, 0.1 mM) produced an 8.7-fold increase in TG activity within 5 min that lasted for 30 min and returned to control levels by 2 h. In contrast, the ACh-induced increase in TG activity in NG, a tissue containing neuronal cell bodies with few synapses, was more gradual and of smaller magnitude, reaching a peak of approximately 2.4 times control by 30 min that was maintained for at least 2 h. In both tissues the ACh-stimulation was effectively blocked by the nicotinic antagonist, hexamethonium (0.1 mM), whereas the muscarinic antagonist, atropine (0.1 mM), partially blocked the ACh effect in SCG and was without effect in NG. Addition of the hyperpolarizing adrenergic agonists norepinephrine (NE, 50 microM), isoproterenol (0.2 mM) or dopamine (0.1 mM) produced an inhibition of TG activity in SCG but had no effect in NG. The inhibitory effects of the adrenergic agonists in SCG were blocked by the beta-adrenergic antagonist, propranolol (10 microM) and alpha 2-adrenergic antagonist, yohimbine (10 microM). A kinetic study revealed that the ACh-induced stimulation of TG activity in SCG and NG was a result of decrease in apparent Km and an increase in Vmax value, whereas the NE-induced inhibition of SCG enzyme activity was a result of an increased Km and decreased Vmax. 45Ca2+ influx into excised SCG or NG was significantly reduced by the application of either ACh or NE. The ACh inhibition was effectively blocked by either hexamethonium or atropine. The NE inhibition was more effectively blocked by yohimbine than by propranolol. These results suggest that the rapid alterations of TG activity in SCG produced by cholinergic and adrenergic neurotransmitters are attributable to the processes of receptor-mediated depolarization and hyperpolarization, respectively, via modulation of nerve-impulse-induced Ca2+ fluxes during synaptic activity.

Effects of depolarizing agents on transglutaminase activity, Ca2+ influx, and protein synthesis in superior cervical and nodose ganglia excised from rats.

Rapid changes in transglutaminase (TG) activity, 45Ca(2+)-influx and [3H]leucine incorporation in superior cervical ganglia (SCG), and nodose ganglia (NG) excised from adult rats were examined following addition of membrane-depolarizing agents veratridine (Ver) or high extracellular [K+]o during aerobic incubation in vitro at 37 degrees C. Addition of KCl (50 mM) stimulated TG activity to a maximal extent (four to six-fold) in SCG and NG after 30 min. Ver (0.2 mM) also increased TG activity in both ganglia after 30 min. Kinetic studies showed that the stimulation of TG activity in both ganglia caused by each depolarization condition was associated with a decrease in Km and an increase in Vmax value. The depolarizing agents Ver and high [K+]o also caused significant increases in 45Ca2+ influx into both ganglia. The Ver-induced increases in TG activity and 45Ca2+ accumulation were antagonized by tetrodotoxin (TTX, 1 microM), a sodium channel blocker. The K(+)-induced increase in TG activity was not blocked by tetraethylammonium (TEA, 20 mM), a potassium channel antagonist, although TEA did block the K(+)-induced increase in 45Ca2+ accumulation. The membrane-perturbing, sialic acid-containing compounds, GM1-ganglioside (GM1, 5 nM) and alpha-sialyl cholesterol (alpha-SC, 20 microM), were moderate inhibitors of the K(+)-induced effects on TG activity and 45Ca2+ accumulation. The sialyl compounds had little effect on Ver-induced accumulation of 45Ca2+ but enhanced the Ver-evoked stimulation in TG activity. These results suggests that the veratridine- and K(+)-induced increases in TG activity occur via modulation of Ca2+ and Na+ channel gating mechanisms that are pharmacologically distinct for each depolarizing agent. The veratridine- and K(+)-induced decrease in [3H]leucine incorporation could be a result of stimulation of TG activity as a consequence of degenerative alterations.

Effects of GM1-ganglioside and α-sialyl cholesterol on amino acid uptake, protein synthesis, and Na+, K+-ATPase activity in superior cervical and nodose ganglia excised from adult rats

We examined the effect of GM1-ganglioside in combination with cholera toxin B, and synthetic alpha-sialyl cholesterol (alpha-SC) on neutral amino acid (tritiated alpha-aminoisobutyric acid, [3H]AIB) uptake, protein synthesis [( 3H]leucine incorporation), and Na+,K(+)-ATPase activity in isolated superior cervical ganglia (SCG) and nodose ganglia (NG) from adult rats after aerobic incubation, usually for 2 h at 37 degrees C in vitro. Cholera toxin B, that specifically masks the oligosaccharide chain of GM1-ganglioside, antagonized the GM1-induced changes in [3H]AIB uptake, [3H]leucine incorporation, and Na+,K(+)-ATPase activity almost completely in SCG, but partially in NG. Although cholesterol itself had little effect on either [3H]AIB uptake and Na+,K(+)-ATPase activity both in SCG and NG, alpha-SC caused considerable reduction of both amino acid uptake and the transport enzyme activity in each ganglia. However, cholesterol was more effective than alpha-SC in decreasing [3H]leucine incorporation in either ganglia. Whereas addition of EGTA markedly reduced either GM1-induced or alpha-SC-induced change in [3H]leucine incorporation into acid-insoluble fraction in both SCG and NG, application of Ca2+ ionophore produced considerable recovery of the protein synthesis from the inhibited level by Ca2(+)-deprivation. ATP and creatine phosphate contents in SCG were elevated by the presence of GM1 or alpha-SC, whereas [3H]AIB uptake and Na+,K(+)-ATPase activity were inhibited, suggesting that utilization for membrane transport was diminished as a result of GM1- or alpha-SC-induced decrease of ATPase activity.

Possible involvement of nitric oxide in carbachol-induced activation of transglutaminase in rat superior cervical sympathetic ganglia

The addition of a muscarinic agonist, carbachol (Carb, 0.1 mM), to a physiological medium markedly increased Ca(2+)-dependent transglutaminase (TG) activity (approximately 10-fold) in isolated rat superior cervical sympathetic ganglia (SCG) following in vitro aerobic incubation for 30 min at 37 degrees C. The Carb-evoked stimulation of ganglionic TG activity was considerably reduced (-51%) in the presence of NG-monomethyl-L-arginine (L-NMMA, 50 microM), a selective inhibitor of nitric oxide (NO) synthase. While the suppressant effect of L-NMMA was completely eliminated by the addition of an excess concentration of L-arginine (0.5 mM), a precursor of NO. These observations imply that Carb-induced TG activation possibly involves NO mediation in SCG tissue. The Carb-induced elevation in ganglionic TG activity was markedly reduced (-84%) at as early as 15 min of incubation in the medium containing hemoglobin (Hb) (20 microM), an agent that scavenges only extracellular NO gas. Thus, it is evident that a large fraction of NO released from inside the neuronal cells to extracellular space could rapidly diffuse back into the same group of cells to induce activation of the tissue TG. Methylene blue (MB), an inhibitor of soluble guanylate cyclase (GC), at 0.5 mM, a concentration which is effective in almost abolishing the Carb-evoked synthesis of cyclic GMP (cGMP), had no effect on ganglionic TG activation induced by Carb. Therefore, an increase in cGMP synthesis mediated by NO might not participate in NO-dependent ganglionic TG activation following the stimulation with Carb.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of axotomy on nitric oxide-dependent cyclic GMP production of rat superior cervical sympathetic ganglia in response to norepinephrine

Cyclic GMP (cGMP) production in superior cervical sympathetic ganglia (SCG) isolated from rats was markedly enhanced (approx. 4.5-fold) by the addition of L-arginine (L-Arg, 100 microM) plus an inhibitor (3-isobutyl-1-methylxanthine) for cGMP hydrolytic enzyme during in vitro aerobic incubation at 37 degrees C for 10 min. This accelerated accumulation of ganglionic cGMP was effectively reversed by approximately 50% when NG-monomethyl-L-arginine (L-NMMA, 50 microM), a compound that inhibits nitric oxide (NO) synthesis from L-Arg, was further added to the medium. These observations imply that cGMP production with possible involvement of a mechanism depending on NO synthesis may be functionally operating in the ganglionic tissue. Application of norepinephrine (NE, 50 microM) with pargyline, a monoamine oxidase inhibitor, to the medium also elevated the ganglionic cGMP level at a magnitude comparable to that shown by L-Arg addition, while co-addition of L-NMMA largely (approx. -60%) eliminated the NE-induced increase in ganglionic cGMP formation. In axotomized SCG one week prior to examination, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, augmented stimulatory effect (more than 8-fold) of NE on cGMP production was seen compared to that caused in unoperated ganglia or in SCG 1 week following denervation, where preganglionic cholinergic nerve terminals were destroyed. When axotomized SCG were transferred to in vitro incubation conditions, addition of an alpha-1 adrenergic antagonist, prazosin (1 microM) to the medium virtually reduced the accelerative effect of NE to less than 25% of the NE-induced cGMP level in the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade effect of nerve growth factor on GM1 ganglioside-induced activation of transglutaminase in superior cervical sympathetic ganglia excised from adult rat

The activity of transglutaminase (TG), a Ca(2+)-dependent enzyme indicating tissue degradation or differentiation, showed in isolated adult rat superior cervical ganglia (SCG) a rapid (within 15 to 30 min) and marked (approx. 5- to 8-fold) increase with the addition of either GM1 ganglioside (GM1, 5 nM), which is rich in synapses, or sialyl cholesterol (SC, 20 microM), a synthetic sialic acid-containing compound, to the incubation medium at 37 degrees C. Under the same incubation conditions, addition of GM1 or SC decreased protein kinase C (PKC) activity (-26% to -39%) in the cytosolic fraction of the SCG, but increased the enzymic activity (+39% to +61%) in the particulate (cell membrane) fraction, suggesting that a sialic acid-containing compound (GM1 or SC) promotes PKC translocation from the cytosol to the membrane in ganglionic neurons. By contrast, addition of a promoting factor for survival of sympathetic neurons even in adulthood, nerve growth factor, (NGF, 0.25 micrograms/ml) to the medium significantly decreased ganglionic TG activity (-43%). This inhibition was completely antagonized by the co-addition of NGF-monoclonal antibody (0.75 microgram/ml). An effective blockade of GM1- or SC-induced stimulation of ganglionic TG activity was seen by further addition of NGF to the medium. Also, NGF almost abolished the translocation of ganglionic PKC activity induced by the sialic acid-containing compounds, although either NGF or 12-O-tetradecanoylphorbol ester (TPA) alone stimulated the cytosolic PKC activity (approx. +30%) in the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)