Biochemical evidence for cholinergic involvement in the Limulus brain

Camp. Biochem. P/q&I.

0306-4492/87$3.00 + 0.00 Pergamon Journals Ltd

Vol. 86C. No. I, pp. 103-110. 1987

Printed in Great Britain

BIOCHEMICAL EVIDENCE INVOLVEMENT IN THE MICHAEL

T.

IVY*

and

FOR CHOLINERGIC LIMULUS BRAIN

JAMES G. ToWNsELt

*Department of Physiology and Biophysics, Health Sciences Center. University of Illinois at Chicago, Chicago, IL 60680, USA and TDepartment of Physiology, Meharry Medical College, Nashville, TN 37208, USA. Telephone: (616) 327-6288 (Receiced

25 Murch 1986)

Abstract-l.

The transport of [‘HIcholine by the corpora pedunculata of the circumoesophageal ring gland (brain) of Limulus polyphemus was studied. Corpora pedunculata slices were incubated individually in Chao’s solution containing 0.01 p M [3H]choline at room temperature (25 k 2 ‘C) and readily accumulated the radiolabel from the extracellular environment. 2. The corpora pedunculata uptake of [‘HIcholine was linear over 60 min. 3. The kinetic analysis indicated the existence of dual uptake systems for choline within the corpora pedunculata, a high affinity choline uptake process (K,,, = 0.54 PM and V,,,,, = 0.037 pmoles/mg/min) and a low affinity process (K,,, = 137 FM and V,,,,, = 6.3 pmoles/mg/min). 4. The high affinity choline transport system was dependent on sodium ions and was inhibited by micromolar concentrations of hemicholinium-3. 5. The pre-exposure of the corpora pedunculata to Chao’s solution containing 90 mM potassium for 15 min resulted in a 24% increase in the velocity of the high affinity choline uptake process (V,,,,, = 0.046 pmoles/mg/min). 6. The 90mM potassium Chao’s pretreatment stimulated a substantial increase in the synthesis of

[‘Hlacetylcholine by the corpora pedunculata. 7. The results suggest that the high affinity pedunculata is associated terminals in this tissue.

with the synthesis

choline uptake process of the transmitter acetylcholine,

INTRODUCTION

within the Limuluscorpora presumably within cholinergic

synthetic enzyme for ACh, and the degradative enzyme, acetylcholinesterase (AChE. EC 3. I. I .7) (Malthe-Sorenssen and Emson, 1976; Townsel et al., 1977). The presence of dual uptake systems for choline has been demonstrated in the abdominal ganglia of Limulus (Maleque et al., 1979). Subsequently, it was shown that within the abdominal ganglia, the [3H]choline taken up via the high affinity process was converted, in substantial amounts, to [3H]ACh, releasable under conditions supportive of transmitter release (Newkirk et al., 1980). Moreover, the Limulus CNS tissue yields a population of synaptosomes with sholinergic properties (Newkirk et al., 1981). A comprehensive study of presumed cholinergic components in different Limulus tissues revealed a probable high affinity uptake process for [3H]choline in the corpora pedunculata, circumoesophogeal ring and cardiac ganglion (Sukumar et cd., 1983). Furthermore, within the corpora pedunculata, [3H]choline taken up via the high affinity process was shown to be converted to [3H]choline releasable under conditions known to support transmitter release. In the present study, we characterized the high affinity uptake of [‘HIcholine in the Limulus corpora pedunculata. We also report on the short term metabolism of [‘HIcholine taken up by the high affinity process in this tissue.

It

has been reported that the uptake of choline into nervous tissue is mediated by two uptake processes designated as high affinity uptake and low affinity uptake systems (Yamamura and Snyder, 1972, 1973; Haga and Noda, 1973). The low affinity choline uptake system (LAChUS) exhibits little sodium (Na+)-dependence and is thought to contribute marginally to the synthesis of acetylcholine (ACh) in cholinergic neurons (Diamond and Milfay, 1972; Haga and Noda, 1973; Suszkiw et al., 1976). The high affinity choline uptake system (HAChUS) found in cholinergic neurons is Na + -dependent and subserves the synthesis of ACh (Haga and Noda, 1973; Yamamura and Snyder, 1973; Kuhar et al., 1975; Murrin et al., 1977). It is generally held that the Na+dependent HAChUS is specifically localized within cholinergic nerve terminals (Yamamura and Snyder, 1972; Kuhar et al., 1973; Simon and Kuhar. 1976). Evidence has been presented that suggests that the HAChUS subserves the synthesis of releasable ACh and may be rate-limiting in the synthesis of ACh (Mulder et al., 1974; Murrin et al., 1977; Barker and Mittag, 1975; Simon et al., 1976). The evidence continues to accumulate in support of ACh as a transmitter in the central nervous system (CNS) of Limulus polyphemus (Garrey, 1942; Schallek, 1945; Stephens and Greenberg, 1973; Townsel et al., 1976, 1977; Maleque and Townsel, 1977; Thomas et al., 1978). The Lit&us abdominal ganglia have been shown to contain substantial levels of choline acetyltransferase (CAT, EC 2.3.1.6), the

MATERIALS

AND METHODS

Ti.wuc preparations Limulus po1yphemu.s(horseshoe

crabs) were maintained

in

moist conditions at an ambient temperature of 4 6 C. Male

103

104

MICHAEL T. IVY and JAMES G. TOWNSEL

and female animals averaging 15-30 cm across the carapace were used in all experiments. The brain including the corpora pedunculata, individual abdominal ganglia as well as the heart with the attached cardiac ganglion was removed from the animal and further dissected in Chao’s solution (Chao. 1933) containing 440 mM NaCI, 9 mM KC1 and j7 mM CaCi, buffered with 10 mM [N-(2-hydroxyethyl)-lpiperazine-ethanesulfonic acid] (HEPES) to pH 7.4 at room temperature (25 + 2’C). Each tissue was weighed and the wet weight was recorded. [‘HICholine uptake studies Corpora pedunculata slices (2-4 mg) approximately 2 x 2 x 1 mm were prepared by microdissection. Individual slices of the corpora pedunculata were incubated separately in 210 ~1 of Chao’s solution containing 0.01 PM [3H]choline (specific activity 77 Ci/mmole). These tissue slices were incubated at room temperature for various time intervals to determine the time course of [3H]choline uptake. Subsequently, each tissue was washed in four Smin changes of Chao’s solution (500~1 volume), blotted and placed in a glass scintillation vial. A I ml volume of tissue solubilizer (Beckman BTS-450) was added to each vial, which was then capped and left overnight at 65-85’C. Ten milliliters of scintillant (Budget Solve) was added to each vial and the radioactive content of each sample measured in either a Beckman LS 3 133P or Packard Tri-Carb 4530 liquid scintillation system. The counting efficiency of each system varied from 20 to 45 % and 40 to 55 % , respectively. To study the kinetics of the [jH]choline uptake, individual corpora pedunculata slices were used. Each tissue slice was incubated for 30 min in 210 ~1 of Chao’s solution containing 0.02-80 PM [‘HIcholine (specific activities 78-0.5 Ci/mmole). Following incubation, the tissue was washed in four 5 min changes of Chao’s solution (500 ~1 volume) and blotted. The radioactive uptake was determined as previously described. Effect of sodium on [‘HIcholine pedunculata

uptake in the corpora

Corpora pedunculata slices were incubated individually for 30min at room temperature in 210 ~1 of either 0.1 or 100 p M [ZH]choline (specific activities 75 and 0.1 Ci/mmole, respectively) containing either a Chao’s solution altered by the replacement of sodium with lithium or a normal Chao’s solution. Osmolarity of the altered Chao’s solution was measured by means of an osmometer, and determined to be essentially the same as that of normal Chao’s solution. Subsequently, each tissue was washed in four 5 min changes of Chao’s solution (500~1 volume) and blotted. The [3H]choline uptake by each tissue slice was measured as described earlier. Effect of inhibitors on [‘HIcholine uptake in the corpora pedunculata Individual slices of the corpora pedunculata were incubated at room temperature for 30 min in 210 pl of 0.1 PM [3H]choline (specific activity 75 Ci/mmole) in either Chao’s solution containing different concentrations of metabolic inhibitors and choline analogs, or normal (control) Chao’s solution. After incubation, the tissue slices were washed in four 5 min changes of Chao’s solution (500~1 volume) and blotted. The radioactive content of each tissue were determined as described. Kinetics of posr-depolarizalion srimulation of uprake in Ihe corpora pedunculara Corpora pedunculata slices were preincubated separately in a 500 pl volume of either 90 mM K + Chao’s or normal (control) Chao’s solution for 15 min at room temperature. Following preincubation, each tissue slice was washed in two 5 min washes of normal Chao’s solution (500~1

volume), subsequently transferred to 200~1 of normal Chao’s solution containing 0.025-O. I pM [?H]choline (specific activities 78-75 Ci/mmole) and incubated for a fixed time period (20 min). After incubation, each tissue was washed in four 5 min changes of Chao’s solution (500 pl volume) and blotted. The radioactive uptake by each sample was measured as previously described. Comparative study of the effect of high potassium preincubation on the synthesis of [‘HIcholine uptake products in Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion High affinity choline uptake has been demonstrated in both the abdominal ganglia and cardiac ganglion. Previous studies in this laboratory suggest that the abdominal ganglia HAChUS is cholinergic in nature (Maleque ef al., 1979; Newkirk et al., 1980). Conversely, the cardiac ganglion HAChUS is clearly non-cholinergic (Ivy and Townsel, 1985). Thus these tissues were used in a comparative elevated K+ pretreatment paradigm. The corpora pedunculata, abdominal ganglia and cardiac ganglion were preincubated separately in 500 ~1 of either 90 mM K+ Chao’s or normal (control) Chao’s solution for 15 min at room temperature. Subsequently, each tissue was washed in two 5 min changes of normal Chao’s solution (500~1 volume). This was followed by the incubation of each tissue in 200 pl of normal Chao’s solution containing 0.1 p M [‘HIcholine (specific activity 78 Ci/mmole) for 120 min at room temperature. After incubation, tissues were washed separately in four 5 min changes of Chao’s solution (500 ~1 volume) and blotted. Then each tissue was separately homogenized using a Teflon homogenizer in 100~1 of electrophoresis buffer (0.47 M formic in 1.4 M acetic acid) containing carrier

amounts of unlabeled ACh, choline (lOmg/ml each) and phosphorylcholine (PCh) (250 mg/ml). Each tissue homogenate was centrifuged at 20,OOOg for 15 min at 4 C. A 20 ~1 aliquot of each supernatant was spotted on Whatman chromatographic paper and subjected to high voltage electrophoresis for lf$2f hr. The electropherograms were dried in an incubator oven and cut into 1 cm strips. Each strip was transferred to a scintillation vial containing 10 ml of scintillant and counted in a liquid scintillation spectrometer. The counting efficiency varied between 40 and 55%. Sources of materials Horseshoe crabs were obtained from the Marine Biological Laboratories, Woods Hole, Massachusetts. [Methyl‘HIcholine chloride (specific activities 77 or 78 Ci/mmole) was purchased from Amersham Corporation. BTS-450 was purchased from Beckman Instruments Inc. Budget Solve was obtained from Research Products International. All other chemicals used were of reagent grade and were purchased from Sigma Chemical Company and other commercial sources.

RESULTS [‘HICholine

uptake

in the corpora

pedunculata

The accumulation of [‘HIcholine by corpora pedunculata slices was linear for 60 min (Fig. 1). The uptake of [3H]choline was subsequently studied over the range of concentrations indicated. A LineweaverBurk plot of the data revealed two distinct uptake components: one with high affinity kinetics and the other with low affinity kinetics (Fig. 2). The K,,, and V,,,,, for the high affinity component were 0.54 FM and 0.037 pmoles/mg/min, respectively. The values for the same parameters of the low affinity component were 137 p M and 6.3 pmoles/mg/min.

Choline uptake by ~irn~~u.~ brain Time Course of r3ti] Cholme Uptake in the Limbs Brain (Corpora pedunculoto)

105

transformation

IO

of the Michaelis-Menten VmnxHA

v, =

0.01 x 10%[3H]

Choline

ls

vmx

1 +

LA ’

is

1

&LA+[~]

Where V, is the total velocity, [S] is the substrate concentration, and the kinetic constants for both the high affinity (HA) and the low affinity (LA) systems are indicated with subscripts. At 0.1 PM ~3H]choline the high affinity process accounted for approximately 67% of the uptake. On the other hand, the low affinity process contributed 95% of the choline uptake at 100 p M [3H]choline. Sodium withdrawal caused a 72% reduction in high affinity choline uptake (Table 1). The low affinity process was not significantly affected by the absence of sodium. Efict of in~jbifor~ on [3H]clzofineuptake in the torpora ~edunc~~~ta

Time (min.)

Fig. I. Time course for the uptake of [3H]choline by the Limulus corpora pedunculata. Slices of the corpora pedunculata were incubated separately in 0.01 PM [-‘HIcholine for various periods of time at 25 &2”C. Values mean ;t SEM of at least three determinations.

x

K mHA+[Sl

equation:

are the

Various studies have provided substantial evidence that the cholinergic HAChUS is sodium-dependent (Simon and Kuhar, 1976; Kuhar and Murrin, 1978; Murrin, 1980). We assessed the effect of sodium on both the low and high affinity components of choline uptake in the corpora pedunculata. The concentrations of choline used were 0.1 and 100 PM. The reiative contribution of the high affinity and low affinity components of transport at these concentrations was calculated by means of the following

The cholinergic HAChUS is reportedly sensitive to a variety of compounds (Kuhar and Murrin, 1978; Fisher and Hanin, 1980). To further characterize the HAChUS in the corpora pedunculata, several metabolic inhibitors and choline analogs were tested. The high affinity uptake process was markedly inhibited by ouabain, a known inhibitor of Na+/K i-stimuiated ATPase, and 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation (Table 2). Similarly, hemicholinium-3 (HC-3), decamethonium and tetramethylammonium, all quaternary ammonium compounds, caused significant inhibition of choline uptake. The maximum inhibition of uptake was achieved with HC-3. Kinetics oboist-depolari~ati~r~ sfim~~~tionof uptake in the corpora peduncz~~~ta It is reputed that the cholinergic HAChUS is coupled to neuronal activity (Barker, 1976; Newkirk

60

10

9-

54 48 42 -

N

36 -

-0 X >

0

30 -

10’

24 LA K, V,,,

0

137 x 106M 6 3 pmoles

mg ml”

1~~S;??10~

Fig. 2. Lineweaver-Burk plot of choline uptake by the corpora pedunculata as a function of choline concentration. Corpora pedunculata slices were incubated with different concentrations of [‘HIcholine for 30 min at 25 + 2°C. Each point represents the mean + SEM of at least three determinations.

106

MICHAEL

T. IVY and

JAMES G. TOWNSEL

Table 1. Intluence of sodium on the uptake of (3H]choline by the Litnulus corpora pedunculata. Slices were incubated in either 0.1 PM or 100 PM [‘H]choline containing either normal (control) or altered (sodium replaced by lithium) Chao’s solution for 30 min at 25 + Z’C. The percent figures in the parentheses represent the percent uptake of [‘H]choline. Values are the mean + SEM for at least three determinations

Tissue

Incubation

Corpora pedunculata

Chao’s

solution

Sodium replaced (440 mM) *Statistically

significant

compared

Choline uptake (pmoles/mg tissue/30 min) 0.1 OM 100pM

medium (control)

0. I29 + 0.004 (l00.00%) 0.036 I_ 0.004* (28.00%)

by lithium to Chao’s

controls:

(P < 0.05, Student’s

72.175 + 15.238 (100.00%) 56.963 _t 12.094 (73.33%) f-test).

Table 2. Erects of inhibitors on the uptake of [‘HIcholine by the Limulus corpora pedunculata. Slices were incubated in 0.1 PM [‘HIcholine containing either normal (control) or inhibitor (one of the several inhibitors indicated at different concentrations) Chao’s solution for 30 min at 25 + 2 ‘C. Values are the mean + SEM for at least three determinations COW.

Substance

(FM)

Chao’s (control) Hemicholinium-3

500 100 IO SO 100 100 100

Decamethonium 2,4-Dinitrophenol Tetramethylammonium Ouabain “Significantly

different

from controls:

N

Choline uptake as % of control (0.1 pM [3H]choii~e)

18 3 3 3 3 3 3 3

100.00 13.33 i 1.52* 23.33 + 4.6S 26.61 + 3.66* 43.33 tt 3.07* 51.20 -f- 5.33* 62.00 + 4.78* 42.00 +2.X*

(P < 0.05, Student’s

et at., 1980; Higgins and Neal, 1982). Moreover, it has been suggested that depolarizing levels of K+ which cause neurotransmitter release result in an activation of this choline uptake system. To determine the nature of this activation of the high affinity

r-test).

uptake process by 90 mM KC Chao’s preincubation, the effect of this pretreatment on uptake was studied at various concentrations of [‘HIcholine. A double reciprocal analysis (Lineweaver-Burk plot) of the data revealed that 90 mM K+ Chao’s preincubation

30 -

A Normal Chao‘s (control) Preincobatlon 049uM K, Vinax 0 032 pmoles mg mm l

20

_

90 mM Potassum Chaos Preincubatlon K“1 0 50 PM Vmu” 0 046 pmoles mg mln*

(15 mln).

(15 mm)

‘S~gml~cantly dlflerent from control (P ( 0 05. Student’s t+st)

s! 0

X

/

>

Fig. 3. Lineweaver---Burk plot of the high affinity [‘HIcholine uptake following pre-exposure to 90mM potassium (K+) or normal (control) Chao’s at choline concentrations varying from 0.025 to 0.1 PM. Corpora pedunculata slices were preincubated for 15 min in either normal or 90 mM in K” Chao’s solution and then incubated with different concentrations of [3H]choiine for 20 min at 25 & 2°C. Key: normal (control) Chao’s preincubation (A). and 90 mM K+ Chao’s preincubation (0). Each point represents the mean i SEM of three determinations.

107

Choline uptake by Limulusbrain resulted in a 24% increase in the V,,,,, of the corpora pedunculata HAChUS (Fig. 3). The K,,, value of the high affinity uptake process was not altered by this pretreatment. Comparative study of the effect of high potassium preincubation on the synthesis of [‘HIcholine uptake products in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion

In the previous studies, the early fate of [‘HIcholine accumulated via the HAChUS was assessed in the Limulus abdominal ganglia, corpora pedunculata and ORIGIN @

*’

cardiac ganglion (Newkirk et al., 1980; Sukumar et al., 1983; Ivy et al., 1985). Subsequent studies were designed to investigate the effect of elevated K+ Chao’s preincubation on the incorporation of [‘HIcholine taken up into radiolabeled metabolites within these tissues. In all three tissues, elevated K+ pre-exposure did stimulate the uptake of [3H]choline (Ivy et al., 1985). The radiochromatographic analysis of the corpora pedunculata extract following elevated K+ pretreatment of the corpora pedunculata revealed at least a three-fold increase in the synthesis of [‘H]ACh within the supernatant fraction (Fig. 4).

Pch

Ach

0

00

CORPORA

Ch

PEDUNCULATA -

t

h

90 mM K+ Chao’s Prrincubolion (IS nun)

ControlNorm01 Chao's Prrincubation

i

1; - ABDOMINAL

(I6 mid

GANGLIA

n

-90 mM K+ Choo’r Prrmcubatmn

6.

ConrrolNormal Premcubatmn

CARDIAC

Choo’r (I5 mid

(15 mid

GANGLION Premcubotion

Preincubation

(IS min)

(IS mid

Fig. 4. Electrophoretic analysis of the effect of elevated potassium preincubation on the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion following incubation in [‘H]choline. High-voltage paper electrophoresis of corpora pedunculata, abdominal ganglia or cardiac ganglion homogenates were run subsequent to pre-exposure to either normal (control) Chaos or 90mM K+ Chao’s solution for I5 min, followed by separate incubation in 0.1 p M [3H]choline in normal Chaos solution for 120 min at 25 & 2°C. Each tissue homogenate was centrifuged at 20,OOOg for 15 min at 4°C and the resultant pellet was counted as previously described. A 10 ~1 aliquot in duplicate of each homogenate supernatant was counted and the average percentage of the total [‘HI (radioactivity as DPM) of each tissue supernatant was calculated. A 20~1 aliquot of each homogenate supernatant was spotted separately. The electropherogram was cut into 1 cm strips and counted using liquid scintillation spectrometry. A 20 ~1 aliquot of electrophoresis buffer containing phosphorylcholine (PCh), acetylcholine (ACh) and choline (Ch) was run to indicate the positions of these standards. The origin is indicated, as well as + and - referring to the direction of cathode and anode, respectively.

108

MICHAELT. IVY and JAMESG. TOWNSEL

Similarly, the prior exposure of the abdominal ganglia to elevated K+ caused a significant increase in the synthesis of [3H]ACh. On the other hand, this pretreatment of the cardiac ganglion did not alter the distribution pattern of supernatant radiolabel in comparison to its control. A quantitative analysis of the amount of radioactivity recovered as [3H]choline metabolites in the corpora pedunculata, abdominal ganglia and cardiac ganglion yielded the following results. When the corpora pedunculata was previously exposed to 90 mM K+, a 257% increase in the synthesis of recoverable [3H]ACh was observed in comparison to its control (Fig. 5). A 165% increase in [3H]ACh synthesis resulted following elevated K+ pretreatment of the abdominal ganglia. On the other hand, when the cardiac ganglion was pre-exposed to 90 mM K+, the synthesis of recoverable [3H]PCh occurred in nearly

0 015

CORPORA

PEDUNCULATA

the same proportion tissue preparations, taken up remained

as the control preparation. In all various amounts of [3H]choline unmetabolized. DISCUSSION

The results reported here demonstrate that the corpora pedunculata of Limulus accumulates choline from the extracellular medium by two distinct uptake processes. The Limulus corpora pedunculata HAChUS is markedly sodium-dependent. This feature is generally ascribed to the HAChUS presumed peculiar to cholinergic terminals. The corpora pedunculata HAChUS is quite sensitive to inhibition by HC-3, a consistent feature reported for the presumed cholinergic HAChUS (Guyenet et al., 1973; Holden et al., 1975; Simon et al., 1976; Smart, 1981; Breer, 1982; Hemsworth et al., 1984). Decamethonium,

0

Ncnnal CM&

G-mfrol)

Premubatmn

I15mm1

* 90 m&4 K+ Chooi Prcmcubotm

m z

l

0.01

(I5 “an)

dtfferent from Control

IP<005,Sfudcnl’s

: F 8 z

Slgn’flconlly

t-test1

0005

is

0

2

=1 9

6

ABDOMINAL

oo,

-

CARDIAC

GANGLIA

GANGLION

E E

Pch

Ach

Ch

Unknowns

Fig. 5. Effect of elevated potassium preincubation on the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion recoverable [‘Hlmetabolites following incubation in [‘HIcholine. High-voltage paper electrophoresis of corpora pedunculata, abdominal ganglia or cardiac ganglion homogenates were run after pretreatment with either normal (control) Chao’s or 90 mM K+ Chao’s solution tbr 15 min followed by separate incubation in 0.1 PM [3H]choline in normal Chao’s solution for 120 min at 25 * 2°C. Each tissue homogenate was centrifuged at 20,OOOg for 15 min at 4°C and the resultant pellet was counted as previously described. A 10 ~1 aliquot in duplicate of each homogenate supernatant was counted and the average percentage of the total [‘HI (radioactivity as DPM) of each tissue supernatant was calculated. A 20 11 aliquot of each homogenate supernatant was spotted separately. The electropherogram was cut into 1 cm strips and the radioactivity was measured using liquid scintillation spectrometry. A 20 ~1 aliquot of electrophoresis buffer containing phosphorycholine (PCh), acetylcholine (ACh) and choline (Ch) was run to indicate the positions of these standards. The origin is indicated, as well as + and - referring to the direction of cathode and anode, respectively. The values are mean * SEM of picamoles of recoverable [3H]/milligram of tissue for three determinations.

Choline uptake by Limuius brain

109

tetramethyIammonium, 2,4-dinitrophenol and ouaREFERENCE bain have been shown to inhibit choline transport in Adamic S. (I 974) Accumulation of choline by the segmental ganglia of leech. Biochem. Pharmuc. 23, 2595-2602. various tissue preparations (Yamamura and Snyder, of synaptosomal high 1973; Adamic, 1974; Simon and Kuhar, 1976; Barker L. A. (1976) Modulation affinity choline transport. Life Sci. 18, 725-732. Maleque et al., 1979; Ivy et al., 1985). In varying Barker L. A. and Mittag T. W. (1975) Comparative studies degrees, these compounds all inhibited choline of substrates and inhibitors of choline transport and uptake in the corpora pedunculata. choline acetyltransferase. J. Pharmac. exp. Ther. 192, An interesting characteristic of the cholinergic 86-94. HAChUS is that its capacity appears coupled to Breer A. (1982) Uptake of [N-Me-‘HI-choline by synnerve impulse activity (i.e. depolarization) (Simon aptosomes from the central nervous system of Locusta and Kuhar, 1975; Barker, 1976; Kuhar and Murrin, m~grutora. J. ~eurobio~. 13, 107-I 1J. 1978). IFZvitro experiments have demonstrated that Chao I. (1933) Action of electrolytes on the dorsal median nerve cord of the Limuius heart, Biol. RUN.mar. biol. Lab., the HAChUS undergoes post-depolarization stimuWoods Hole 64, 3.58381. lation (Murrin er al., 1977; Murrin, 1980; Higgins and Diamond I. and Milfay D. (1972) Uptake of [‘HImethyl Neal, 1982). Pre-exposure to depolarizing levels of choline by microsomal, synaptosomal, mitochondrial and potassium resulted in a significant increase in the V,,,,, synaptic vesicle fractions of rat brain The effect of of the corpora pedunculata (Fig. 3). Furthermore, hemicholinium. /. Neurochem. 19, 18991909. not only did elevated K’ pre-exposure stimulate Fahrenbach W. H. (1979) The brain of the horseshoe choline accumulation, it stimulated a simultaneous crab (Limufus pu~yphemu~) III. Cellular and synaptic organisation of the corpora ~dunculata. Tissue Cell II, increase in the efficiency of ACh synthesis (Fig. 5). 163-200. In the absence of elevated K+ pre-exposure. approxiFisher A. and Hanin I. (1980) Choline analogs as potential mately 55% of the [3H]chohne taken up was contools in developing selective animal models of central verted to [‘H]ACh. Following exposure of elevated cholinergic hypofunction. Life Sci. 27, 1615-1634. K+. the tissue incorporated approximately 73% of Garrey W. E. (1942) An analysis of the action of acetylthe transported [3H]choline into [‘H]ACh, a 33% choline on the cardiac ganglion of Limulus polvphemus. increase in the efficiency ACh synthesis. The combinAm. J. Phvsiol. 136, 1822193. ation of a 126% increase in choline uptake and a Guyenet P., Lefresne P., Rossier J., Beaujouan J. C. and 33% increase in efficiency of its conversion results Glowinski J. (1973) Inhjbition by he~i~holinium-3 of in an approximate three-fold increase in [3H]ACh. ~‘4Clacetvlcholine svnthesis and r3Hlcholine high-affinity uptake in rat striatal synaptosomes: Molec. Pharmac. 4, These results clearly establish that the HAChUS of 630-639. the corpora pedunculata is capable of generating a significant increase in this tissue’s ACh content in Haga T. and Noda H. (1973) Choline uptake systems of rat brain synaptosomes. Biochim. biophys. Acfa 291, response to depolarization. This property of the 564-575. corpora pedunculata HAChUS suggests that cholinHemsworth B. A., Shreeve S. M. and Veitch G. B. A. (1984) ergic nerve terminals are present in this tissue. Pharmacological actions of some cyclic analogues of Earlier reports have demonstrated the presence of choline. Br. J. Pharmac. 81, 685-692. both CAT and AChE activity within the Limuhs Higgins A. J. and Neal M. J. (1982) Potassium activation of corpora pedunculata (Emson et a!.. 1974; Sukumar et [~H]choIine accumulation by isolated sympathetic ganglia al., 1983). In addition, on the basis of ultrastructural of the rat. &. J. Pharmac. 77, 573-580. Ivy M. T., Sukumar R. and Townsei J. G. (1985) The studies of synaptic profiles within the Limulus corcharacterization of a sodium-dependent high affinity pora pedunculata, Fahrenbach (1969) has suggested choline uptake system unassociated with acetylcholine that ACh is a neurotransmitter within this tissue. The biosynthesis. Camp. Biochem. Phyriol. 81C, 351-357. major product of [“HIcholine accumulated by the Ivy M. T. and Townsel J. G. (1985) High affinity choline corpora pedunculata HAChUS is [‘H]ACh (Ivy et al., uptake and choline metabolism in cholinergic and non1985) and this 13H]ACh is released by elevated K+ cholinergic tissues. Abstr. Annu. Minority Biomedical under conditions known to support the selective Research Support Symposium, 73. nerve terminal release of transmitter (Sukumar et al., Kuhar M. J., DeHaven R. N., Yamamura H. I., Rommel1983). The results presented herein are consistent spacher H. and Simon J. R. (1975) Further evidence for cholinergic habenuiointerpeduncular neurons: Pharmacowith the view that the high affinity uptake process logic and functional characteristics. Bruin Res. 97, for choline in the Limulus corpora pedunculata is 2655275. reflective of cholinergic transmission in this tissue. Kuhar M. J. and Murrin L. C. (1978) Sodium-dependent Both the well described HAChUS within cholinergic high affinity choline uptake. J. Neurochem. 30, 15-21. nerve terminals and the corpora pedunculata Kuhar M. J., &thy V. H., Roth R. H. and Aghajanian HAChUS are sodium-dependent, inhibited by microG. K. (1973) Choline: Selective accumulation by central molar concentrations of HC-3, and are significantly cholinergic neurons. J. Nemo&em. 20, 581.--593. stimulated in the wake of prolonged depolarisation. Maleque M. A., Newkirk R. F. and Townsel J. G. (1979) In sum, our findings are corroborative of the Kinetics of [3H]choline uptake in abdominal ganglia of Limulus polvphemus. Biochem. Pharmac. 2%. 9855990. suggestion that there are chohnergic terminals and Maleque M. A.-and Townsel J. G. (1977) ‘H-Choline uptake hence chohnergic transmission within the corpora in the abdominal ganglia of Limulus pol,vphemus. Neuropedunculata of Limulus. L

Acknowledgements-This work was supported by a BRSG grant from the University of Illinois at Chicago and a MBRS Grant No. RR-08037 from Meharry Medical College.

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