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Biomedical subjects

E Giacobini

Publications and source records attributed to E Giacobini.

At least 127 records · Page 7Linked to original sources

Uptake of piperidine and pipecolic acid by synaptosomes from mouse brain.

Piperidine is actively transported into the synaptosomal fraction of adult mouse brain. The transport mechanism appears to be Na+ independent but is temperature dependent and sensitive to ouabain. Analysis of kinetic experiments indicates only a "low-affinity" transport system to be present. By contrast the uptake of D,L-[(3)H]pipecolic acid at a concentration of 4 X 10(-7)M was temperature and Na+ dependent, ouabain sensitive, and revealed a two-component system with a Km = 3.9 plus or minus 0.17 X 10(-6)M, Vmax = 129 plus or minus 6 pmol/mg protein/3 min for the "high-affinity" system and a Km = 90.2 plus or minus 4.3 X 10 (-6)M, Vmax = 2.45 plus or minus 0.19 nmol/mg protein/3 min for the "low-affinity" system. Compounds structurally related to pipecolic acid such as glycine, L-proline, 4-amino-n-butyric acid, and 5-amino-n-valeric acid showed an inhibitory effect on uptake at a concentration of 10(-4)M. The demonstration of biosynthesis of pipecolic acid in mouse brain and the presence of a "high-affinity" sodium-dependent uptake system suggest a physiological role of this substance in the central nervous system.

Animals↗

Norepinephrine uptake in aging adrenergic nerve terminals.

Norepinephrine (NE) uptake has been studied in the adult (3-6 months) and aging (5 yr) chick iris, in which there is a discrete population of norepinephrine containing nerve terminals. although total accumulation of 3H-NE in the iris does not change with age, there is a decline in Na+-dependence, temperature-sensitivity, ouabain-and inhibitor -sensitivity of uptake. The results indicate either a loss of active, carrier-mediated NE uptake during aging, or a change in the biochemical and pharmacological characteristics of this uptake. changes in the composition of the organ and in the sites of accumulation of NE are considered.

Aging↗

Development and aging of cholinergic synapses. II. Continuous growth of acetylcholine and choline levels in autonomic ganglia and iris of the chick.

We have followed the pattern of variation in the endogenous levels of acetylcholine (ACh) and choline (Ch) in sympathetic (lumbar) and parasympathetic (ciliary) ganglia, and in the iris of the chick from 1 to 7 years of age. Levels of ACh and Ch seem to undergo a continuous process of growth which is initiated in the first week of embryonic development and continues into adulthood up to 2 years of age. The early increase in ACh and Ch levels are most marked in the ciliary ganglion. In the ciliary ganglion, morphological and biochemical observations show that this period corresponds to one of 'continuous synaptic growth'. In a second period starting around 2 years, which can be defined as one of 'synaptic regression', a phase of progressive and marked decrease in ACh and Ch levels is visible in all three organs examined. In all three organs Ch levels parallel rather well the ACh levels, but the Ch/ACh ratio progressively decreased from 1 to 7 years from 3.5 to 1.5 (ciliary ganglia and iris) and from 2.5 to 1 (sympathetic ganglia). The low levels of ACh reached at 7 years in the three organs have clear implications with regard to physiology of aging cholinergic synapses.

Acetylcholine↗

Development and aging of cholinergic synapses. III. Choline uptake in the developing iris of the chick.

Choline (Ch) uptake in the iris of the chick has been characterized from 4 1/2 days of incubation to 1 year of age, in conjunction with an electron microscopic ultrastructural study of the innervation of the iris. Uptake has been shown to constitute an early and reliable biochemical marker for the presence of innervation in target organs. The characteristics of Ch uptake, including Na+ and temperature dependence and inhibitor sensitivity, change during development, and the Vmax increases steadily while the Km does not vary significantly during the period of study. The relationships among uptake and other cholinergic functions also change with development of the iris. Acetylcholine (ACh) and Ch uptake but not Ch levels appear to develop together, while cholinergic enzyme activities follow a developmental course more closely related to neurotransmission. The significance of these findings in relation to proposed regulatory relationships in cholinergic biosynthetic mechanisms in discussed.

Aging↗

Development and aging of cholinergic synapses. IV. Acetylcholinesterase and choline acetyltransferase activities in autonomic ganglia and iris of the chick.

Age-related changes in the activities of choline acetyltransferase an acetylcholinesterase in avian sympathetic ganglia, ciliary ganglia and iris have been determined throughout the posthatching life span. Kinetic characteristics of these enzymes have been analyzed in the nerve terminals in the iris from early embryonic stages to the latest periods of life. Enzyme activities and kinetics have been related to each other and also to the endogenous levels of acetylcholine. The regulation of the biosynthetic mechanism for acetylcholine through choline uptake and choline acetyltransferase activity in the developing nerve terminal have been examined. The activities of both enzymes in all tissue are reduced at later ages, which is referred to declines in Vmax, as the Km values do not vary significantly at any age. Declines with age are most pronounced in the iris, both in choline acetyltransferase activity and Vmax and in acetylcholine levels, supporting our view of the peripheral autonomic nerve terminal as a major locus for the effects of aging on the peripheral nervous system.

Acetylcholinesterase↗

Molecular forms of cholinesterases in cerebrospinal fluid, blood plasma, and brain tissue of the beagle dog.

In the present study we have investigated the activity of AChE and BuChE in the cisternal CSF of the Beagle dog and have outlined the characteristics of the molecular forms of both enzymes. The same enzymes were also investigated in samples from blood plasma and brain tissue. It is concluded that AChE in CSF originates from the spinal cord and brain tissues as a result of some secretory process. A combined origin from both blood plasma and brain tissue appears to be probable for the BuChE of the CSF.

Acetylcholinesterase↗

Normal distribution and denervation changes of neurotransmitter related enzymes in cholinergic neurones.

1. The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in adult pigeon ciliary ganglia, in the post-synaptic ciliary and choroid nerves, and in ciliary nerve iris terminals isolated from control birds and from animals from which the oculomotor nerve was previously transected. Enzyme activity levels were also measured in the iris terminals after surgical section of the ciliary nerves. From differences in enzyme activity between control and 3-day denervated tissues, the localization of CAT and AChE in pre- and post-synaptic elements of the ganglia and at the iris neuromuscular junctions was estimated. The fate of the preganglionic nerve terminals after denervation was investigated by electron microscopic examination of ganglia after surgical section of the oculomotor nerve.2. The CAT activity in the ganglion was distributed as follows: 60% in presynaptic elements, 31% in cell somas, and 9% in intraganglionic post-synaptic axons; in the iris junctions, 98% of the activity was present in the ciliary nerve terminals. For AChE: 20% was present in the preganglionic terminals, 69% in ganglion cell somas and the remaining 11% in post-ganglionic axons; at the neuromuscular iris junctions, 20% was found in the ciliary nerve terminals and 80% in the iris striated muscle.3. The first changes in the fine structure of the nerve terminals were observed 14 hr after surgery, and by 24 hr marked alteration of the synaptic structure were clearly recognized. No preganglionic endings were found in 3 day-old denervated ganglia.4. There was a positive correlation between CAT activity in the control iris nerve terminals and in ganglia. After denervation, when the activity of the enzyme decreased in ganglion cell somas, there was a corresponding decrease in the post-synaptic nerves. These two findings suggest that CAT slow axoplasmic transport is related to its perikarial concentration.5. There was a 60% reduction of CAT activity in the post-synaptic elements, assayed in the 10-day denervated ganglia, which was accompanied by a 30% decrease in activity in the iris nerve terminals. Similarly, post-synaptic AChE decreased approximately 30% in the ganglion and approximately 30% in the iris 10 days after section of the oculomotor nerve. At the same time, CAT activity also decreased in the nerve trunks, 70% at the ciliary nerve and 40% at the choroid; for AChE there were smaller changes.6. In contrast to CAT and AChE, there were no differences in ganglionic protein content, or lactate dehydrogenase (LDH), co-enzyme A (CoA) and monoamine oxidase (MAO) levels between short-term (3 days) and long-term (10 days) denervated ganglia.7. The later decrease of CAT and AChE activity in the cell somas, axons and nerve terminals after long-term preganglionic transection suggests that the activity of these enzymes is regulated across the synapses. It is postulated that the AChE regulation is part of a general ;trophic interaction' between neurones, but that the trans-synaptic modulation of CAT is specific for cholinergic cells.

Acetylcholinesterase↗

Development and aging of cholinergic synapses. I. Endogenous levels of acetylcholine and choline in developing autonomic ganglia and iris of the chick.

Acetylcholine (ACh) and choline (Ch) levels were measured by means of a sensitive micromethod in developing irises, ciliary and sympathetic ganglia of the chick, starting at 5--7 days of incubation (DI) up to 1 year of age. The neurotransmitter is present in all three organs at relatively low levels (1--10 pmol) from 5 DI (iris) or 7 DI (ciliary and sympathetic ganglion). This is followed by a rapid and sustained 9-fold increase in all three tissues up to 14 DI. Thus, it appears that low levels of ACh may be sufficient for neurotransmission to occur in the primitive ciliary ganglion and iris. After hatching, total ACh levels continue to increase up to 1 year of age in the iris and ciliary ganglion and up to 3 months in the sympathetic ganglion. The increase may depend on either an increased functional demand for the neurotransmitter or an increased number of preganglionic terminals. In general Ch levels parallel closely the levels of ACh in each organ throughout development. It is concluded that ACh and Ch are present since the earliest detectable stages of development in the ganglia and iris, and their first increase seems to be correlated to the phase of innervation of the organs. The subsequent increase probably correlates to synaptogenesis. The close relationship between Ch and ACh levels indicates a regulatory role of Ch for ACh synthesis during neuronal development.

Acetylcholine↗

Acetylcholinesterase activity in ventricular and cisternal CSF of dogs: effect of chlorpromazine.

Acetylcholinesterase (AChE) activity in dog plasma is significantly higher than in either ventricular or cisternal CSF. However, since protein levels in plasma are about 100-fold higher than in CSF, the specific activity of AChE is lower in plasma than in CSF. Acetylcholinesterase activity in plasma represents only 22% of total cholinesterase (ChE) activity, while preliminary findings indicate that in ventricular CSF it is 50-60%. Acetylcholinesterase activity in ventricular CSF is significantly lower than in cisternal CSF. Chlorpromazine (10 mg/kg, intravenous), a drug which increases acetylcholine turnover, increased AChE-specific activity in all dogs. Our results support the hypothesis of a neuronal origin of AChE activity in CSF.

Acetylcholinesterase↗

Time course of appearance of alpha-bungarotoxin binding sites during development of chick ciliary ganglion and iris.

The binding of [125I]alpha-bungarotoxin (ABTX) to homogenates of ciliary ganglia and irises from embryonic and posthatching chickens has been examined. Specific, high-affinity binding was found in both tissues [KD (iris) equals 2.5 nM; KD (ganglion) equals 2.7 nM]. Binding is saturated above 10 nM toxin concentration and is inhibited by low concentrations of the nicotinic antagonist d-tubocurarine. The binding may be associated with a nicotinic cholinergic receptor in both tissues. The amount of binding in the iris begins to increase soon after functional innervation is first observed, at 12 days of incubation (d.i.), and continues to increase up to four months after hatching (a.h.), the oldest age tested. In contrast, ABTX binding in the ciliary ganglion increases fourfold between 7 and 11 d.i., after which the amount of binding remains unchanged up to four months a.h. When compared to the development of choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) activities in the ganglion and iris, ABTX binding follows a pattern similar to that of AChE activity. The largest increases in ChAc activity occur later than those of the postsynaptic markers. After 16 d.i. there are approximately 3 x 10(6) toxin molecules bound per neuron in the ciliary ganglion.

Acetylcholinesterase↗

Molecular forms of acetylcholinesterase in the ciliary ganglion and iris of the chick. Developmental changes and effects of axotomy.

Four molecular forms of acetylcholinesterase (AChE) have been observed in extracts from ciliary ganglion and iris by gel electrophoresis followed by Koelle's reaction and densitometry on gels. Forms I, II, and III (order of anodic migration) are present in the 'soluble fraction' (SF) while forms II, III, and IV appear in the 'membranous fraction' (MF). 10 days after postganglionic axotomy in young chickens, the relative proportions of the membranous forms were changed in both the ciliary ganglion and iris, with a marked reduction of form IV occurring. A characteristic evolution of both the soluble and membranous forms of AChE was evident through different developmental stages (10th day of incubation; 2nd, 11th and 56th day posthatching). In the ciliary ganglion the faster components were reduced in favor of form IV. Conversely, the relative proportion of the faster components was increased in the iris. The observed changes in the AChE forms are compared with changes in the structure and function of these organs known to occur in the conditions under study. Evidence is presented which suggests a junctional localization for the form IV in both the ciliary ganglion and iris.

Acetylcholinesterase↗

The effect of a single dose of reserpine administered prior to incubation on the development of tyrosine hydroxylase activity in chick sympathetic ganglia.

A single dose of reserpine administered into the yolk sac of chicken eggs prior to incubation produces two distinct periods of significant increase in tyrosine hydroxylase (TH) activity over controls. The first period is 21 days of incubation (55%) and the second is between day 14 and 30 after hatching (a.h.) (69%). Cholineacetyltransferase (ChAc) and dopadecarboxylase (DDC) are not modified in the two periods of increased TH activity. Reserpine had no effect on cholinergic parasympathetic synapses and neurons in the ciliary ganglion, as judged by ChAc activity. When reserpine was acutely administered in three different posthatching periods only the injection at the latest period (days 26 and 27) caused a significant (38%) increase in TH activity at day 30. Postsynaptic nicotinic receptors were blocked selectively by injecting chlorisondamine in the chick starting at hatching for one week. The administration of chlorisondamine almost completely abolished the reserpine induced increase of TH activity at day 15 a.h. The present results support the view that the development of enzyme activities specifically related to neurotransmitter biosynthesis in chick autonomic ganglia is regulated not only by transsynaptic influences but also by regulatory inputs originating in the periphery.

Animals↗

Developmental variations of tyrosine hydroxylase and acetylcholinesterase in embryonic and post-hatching chicken sympathetic ganglia.

The developmental variations of tyrosine hydroxylase (TH) and of acetylcholinesterase (AChE) were studied in embryonic and post-hatching chicken sympathetic ganglia. Different levels of TH activity were found in two different flocks of White Leghorn chicken, which are probably dependent on genetic differences. These enzymatic differences, however, do not become apparent before hatching and may indicate a combined effect of genetic variation and functional demands. During the period of incubation, TH activity is characterized by a pronounced and steady increase from the twelfth day of incubation up to day 2 after hatching. This corresponds to a period of intense maturation of the sympathetic neuron. In the period following hatching, the 'fourth day fall phenomenon' previously described by us for DOPA decarboxylase (DDC), dopamine-beta-hydroxylase (DBH), and monoamine oxidase (MAO) is not seen in the TH curve. Instead, TH activity tends to remain constant between days 2 and 14 after hatching (ah). Both ganglionic protein and weight remain constant in this period, indicating a phase of general pause in protein synthesis. AChE activity increases steadily from the eighth until the twenty-first day of incubation. A sudden and significant drop in AChE activity was found at day 2 ah followed by a period of rapid increase at day 3 ah and a levelling of activity up to day 30 ah. Comparing the present variations to those observed in our previous studies on DBH, a temporal relationship between TH and DBH activity is observed during the phases of synaptogenesis and maturation but not during the phase of intense functional activity. Our results strongly suggest that before hatching in chick embryo sympathetic ganglia, the cholinergic presynaptic terminals play a role in regulating the development of the adrenergic neurons. In the period following hatching, however, the DBH and TH levels in cell bodies seem to be principally regulated by the functional activity. This results in depletion of DBH, but not TH, through liberation along with the neurotransmitter at the periphery. Depletion of DBH at the terminals may result in increased transport and thereby depletion in the cell body. This mechanism is probably responsible for the difference in the profiles of activity of DBH and TH in the cell bodies observed in the first week after hatching.

Acetylcholinesterase↗

Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation.

1. In chick ciliary ganglia and irises, cholineacetyltransferase (ChAc) and acetylcholinesterase (AChE) activities were measured from the fifth day of incubation until 1 week after hatching. The changes in enzyme activity were correlated in time with previous electrophysiological and morphological findings of synapse formation in these tissues. 2. At Stage 26 (Hamburger & Hamilton, 1951; before synapse formation in the ganglia) low activities of ChAc (12 +/- 4 [mean +/- S.E.] p-mole of ACh synthesized/hr) were measured in the iris nerve terminals, indicating that ganglion cells are biochemically differentiated, immediately after cell migration is completed. The specific acitivities of ChAc and AChE rose during development and these increases were closely related to the onset and maturation of ganglionic and iris synaptic transmission. These increases in enzyme activities can be used in cholinergic synapses as an index of synapse formation. 3. The 200-fold specific increase of ChAc in iris nerve terminals which occurs at Stage 34 probably reflects an increase in synthesis of the enzyme in ganglion cells and suggests that the formation of the iris neuromuscular junction triggers the enzyme induction. It is implied that the cell responds to a signal ascending the axon from the terminal. 4. The initial increase of AChE specific activity in the ganglion occurs after transmission is established in all cells between Stage 30 and 34 and is mainly due to enzyme synthesis by the ganglion cells. In the iris there is a twofold increase in specific activity after the formation of neuromuscular junctions which probably reflects enzyme induction in the muscle subneural region. It is concluded that the specific induction of AChE in post-junctional cells is due to an influence of the prejunctional element. 5. During synaptic formation in the ciliary ganglion, reciprocal interactions between the neurones and their targets result in the induction of ChAc in the prejunctional elements and AChE in the post-junctional cells.

Acetylcholinesterase↗