Acta physiol. scand. 1979. 105. 108-113 From the Department of Pharmacology, Linkoping University. Sweden
Synthesis of 3H-acetylcholinein the rabbit lacrimal gland and its release by electrical field stimulation BY JARLE. S. WIKBERG
Received 17 May 1978
Abstract WIKBERG, J. E. S. Synthesis of 3H-acetylcholine in the rabbit lacrimalgland and its release by electrical field stimulation. Acta physiol. scand. 1979. 105. 108-1 13. Electrical field stimulation was applied to the rabbit lacrimal gland during incubation in uitro with 3Hcholine, after which the efflux of tritiated metabolites was studied. In the presence of physostigmine 3Hacetylcholine was released into the incubation medium when the gland was stimulated electrically. The identity of the 3H-acetylcholine synthesized by the preparation was established by its identical ion exchange chromatographic properties to native acetylcholine. The tissue contents of 3H-acetylcholine and 3H-choline were 14.4 and 3.6"/,, respectively, of the total activities found in the gland. This study demonstrates that the rabbit lacrimal gland is innervated cholinergically. It is suggested that the method developed might be of value for studying effects of drugs on cholinergic neurotransmission in this organ.
The main secretory innervation of the mammalian lacrimal gland is thought to be cholinergic (Demtschenko 1872, Tepliachine 1894, Arenson and Wilson 1971). In previous studies of the cholinergic neurotransmission in this organ measurement of tear secretion has generally been used for indirect determination of acetylcholine secretion from the nerves. Recently a radiochemical method for studying acetylcholine release from isolated guinea pig ileum was described (Wikberg 1977). In the present work a similar approach was used to study the release of acetylcholine from isolated rabbit lacrimal glands. Electrical field stimulation was applied to the organ during incubation with 3H-choline, whereafter the release of 3H-acetylcholine synthesized by the organ could be examined.
Methods Rabbits of either sex were killed by a blow on the neck and exsanguinated. One of the lacrimal glands was rapidly excised through the caudal supraorbital notch and its capsule was carefully dissected away with forceps. The organ (weighing 114k 11 mg), was mounted in a 4 ml water-jacketed perspex chamber containing Krebs solution (Wikberg 1977) with eserine sulphate 3 x M. The solution was kept at 37°C and gassed with a mixture of 0, (95 X) and CO, ( 5 %). The lacrimal gland was incubated with 9.3 x lo6 Bq (becquerel, dps) of 3H-choline (methyl 3H-choline chloride 3.7 x loL1Bq/mmole, Radiochemical Centre, Amersham, England) at a final concentration of 1 x lo-" M, for 1 h. During the incubation the organ was stimulated electrically by 0.1 ms, 320 mA rec-
108
ACH SYNTHESIS IN RABBIT LACRIMAL GLAND
--
Fig. 1. Release of tritiated metabolites from the lacrimal
1
109
Field stimulation 10Hz c
20-
S.E. of 5 expts.
Time (mid tangular pulses a t 0.1 Hz by means of two 2 0 1~mm platinum electrodes mounted 10 mm apart on each side of the organ, using a Grass SD9 stimulator and a home-constructed timer. After the incubation the electric stimulation was stopped and the organ was washed with Krebs solution at intervals of 5 min for 45 min, after which 4 ml fractions were collected for analysis. Rarliochemical assays The method described previously (Wikberg 1977), with some minor modifications, was used, to separate the radioactivity of the samples into 3 fractions: 3H-acetylcholine, ,H-choline and a fraction which remained unprecipitated by ammonium reineckate (UPF). In order to improve the paper chromatographic separation of acetylcholine and choline, a descending rather than the ascending system with acetone/water/lO N HCI 100:20:1 (by volume) was used. The chromatograms were allowed to run for 10 h, using the "durchlauf" technique. To facilitate the identification of carrier choline and acetylcholine the spots were developed by spraying with an iodoplatinate reagent (Walach et al. 1967). By this method the absolute distance between the spots was 3 cm. In order to elute the radioactivity from the paper and to decolorise the iodoplatinate reagent, a mixture of 3 ml ethanol/0.5 M NH, 1 : 1 (v/v) was used. These samples could be counted after addition of 1nstagelO (Packard Instrument Company Inc.) as described previously. After the expt. the lacrimal gland was frozen in Frigen solid CO, and kept at - 80°C until analysed. The tissue was homogenized with a glass homogenizer in 0.4 M HCIO, (1 0.5 ml) at 0°C and then centrifuged a t 10 000 x g for 30 min. The pellet was washed once with 1 ml of the HCIO, solution. Th e pooled supernatant was diluted to 25 ml and 1 ml fractions were taken for analysis as described above for the bathing solution. The pellet was dissolved by adding 2 ml Soluene 350@ (Packard Instrument Company Inc.) and counted in a Packard Tri Carb 3375 liquid scintillation spectrometer after addition of a toluenebased scintillator.
+
Ion exchange chromatography
An approach similar to that described previously was used (Wikberg 1977). Columns measuring 4.4 x 450 mm were packed with Amberlite C G 50 11 (200-400 mesh) equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. The samples applied on the column were eluted with 5 ml H,O followed by the phosphate buffer a t a flow rate of 5 ml/h. In order to determinate the retention volume of acetylcholine, 1.3 x lo3 Bq of 14C-acetylcholine (A~etyI-l-~~C-acetylcholine iodide 8.5 x I O'O Bq/mmole, NEN) was added to the sample and the radioactivity was separated from the tritium activity by measurement at separate channels in the
110
JARL E. S. WIKBERG
I
!'
Fig. 2. Ion exchange chromatography of the reineckate-precipitated fraction in the washing solution from the lacrimal gland. The medium was collected during electrical field stimulation a t 10 Hz and the quaternary ammonium compounds were precipitated by addition of ammonium-reineckate in the presence of carrier acetylcholine and choline. After batch treatment with Biorex 9 in water and addition of 1.3 2 lo3 Bq, 14C-acetylcholine as a marker, the sample was applied on the column, followed by elution with 5 ml water and 0.1 M phosphate buffer, pH 7.0. Insert: Correlation between the 3H-a~tivityin the first peak and the 14C-activity of the 14C-acetylcholine marker (r = 1.000).
liquid scintillation spectrometer. The data obtained were corrected for quenching and for overlap between channels, using an internal standard and a double isotope computer programme written for HP9830. The counting efficiency was 15 "b for 3H and 53 "b for 14C.
Results During unstimulated conditions almost no tritiated acetylcholine was released from the organ. After the 45-min washing period when collection of fractions was started, only 2% of the radioactivity released to the bathing solution consisted of 3H-acetylcholine. Most of the activity at this time consisted of UPF (79%), followed by 3H-choline (19%). Electrical field stimulation with 1.0 ms pulses at 10 Hz induced a 21-fold increase in the release of 3Hacetylcholine. The release of acetylcholine was not maintained at that level but decreased to half its maximal value in approximately 10 min, despite continuous electrical stimulation. The electrical stimulation also induced a two-fold increase in the release of 3H-choline, and during the subsequent stimulation periods this release showed a smaller tendency to decline than that of 3H-acetylcholine. The release of UPF also increased approximately 2-fold on electrical stimulation (Fig. I). The identity of 3H-acetylcholine released during electrical stimulation was verified by ion exchange chromatography. After application of the reineckate precipitated fraction on the column two peaks of radioactivity appeared. The first peak showed excellent correlation to the marker 14C-acetylcholinepeak (correlation coefficient 1 .OOO) (Fig. 2).
ACh
SYNTHESIS IN RABBIT LACRIMAL G L A N D
111
TABLEI. Tritiated metabolites present in the lacrimal gland at the end of the expt. The radioactivity was separated into 4 fractions: the ammonium reineckate unprecipitated fraction (UPF), acetylcholine (Ach), choline (Chol) and the perchloric acid insoluble fraction (Pellet). The table gives the mean S.E. of 5 expts. x Bq
UPF
Ach Chol Pellet
* *
7.55 1.02 2.85k0.35 0.72 & 0.07 8.68 1.44
% 38.1
14.4 3.6
43.8
When the expt. started the tissue content of tritium was 2.18 10.21 y lo5 Bq. This amount corresponded to 23.4% of the total activity of 3H-choline added to the incubation medium at the start of the expt. During the course of the expt. a total of 1.99 k0.60 x lo4 Bq of tritium was released from the lacrimal gland. The secretion of 3H-acetylcholine was 1.22 k 0.10 x lo3 Bq, of 3H-choline 2.93 k0.30 x lo3 Bq and of UPF 1.64 rtr0.77 x lo4 Bq. At the end of the expt. a considerable amount of 3H-acetylcholine was present in the tissue but the 3H-choline content was low. The largest amounts of activity, however, were found as UPF and in the PCA-insoluble fraction (Table I).
Discussion The innervation of the mammalian lacrimal gland has been studied by histological techniques in several earlier studies. Ichikawa and Nakajima (1962) showed that the lacrimal gland of the rat contains unmyelinated nerve fibres in the inter- and intra-lobular connective tissues. Electron microscopic studies on lacrimal glands of monkeys have indicated that parasympathetic nerve fibres originating in the sphenopalatine ganglion innervate interstitial and parenchymal tissues (Ruskell 1969). The arteriole of the monkey lacrimal gland may receive both cholinergic and sympathetic nerves (Ruskel 1967, 1969). Histochemical staining for cholinesterase in the human lacrimal gland has revealed choline esterase activity in myoepithelial cells and nerve fibres surrounding the acinar cells (Mizukawa ef al. 1962). The present study demonstrates that the rabbit lacrimal gland is capable of accumulating tritium when incubated with 3H-choline. It may be estimated that a passive uptake of choline of about 5 % of the amount in the incubation medium will take place under the experimental conditions used. The high uptake observed (23.4 % of the given amount) therefore suggests that an active mechanism for choline transport into the tissue is present. This transport system could be located in nervous tissue as well as in glandular cells. Active uptake of choline into nervous tissue is well documented (Pert and Snyder 1974, Simon et al. 1976, Wikberg 1977), but other cells such as human red blood corpuscles may have transport systems for choline accumulation (Clement and Colhoun 1975). In this work it was found that the rabbit lacrimal gland is capable of synthesizing 3Hacetylcholine from 3H-choline. The chemical identity of 3H-acetylcholine seems to be certain, since the metabolite showed properties identical to those of native acetylcholine in two different chromatographic systems. The basal release of 3H-acetylcholine into the in-
I12
JARL E. S. WIKBERC
cubation medium was almost negligible. This contrasted markedly with the results obtained from guinea pig ileum (Wikberg 1977), where the unstimulated release was much higher and showed spontaneous fluctuations. Electrical stimulation, with stimulation parameters indicating that selective stimulation of nervous tissue was induced, resulted in a large increase of the release of 3H-acetylcholine from the lacrimal gland. The results of these experiments may be taken as fairly clear evidence of the presence of cholinergic innervation in this tissue. The tissue content of 3H-acetylcholine was more than 20-fold larger than the amount of 3H-acetylcholine released during the electrical stimulation. In spite of this the release of 3Hacetylcholine declined rapidly after the initial large release. This observation might indicate that different compartments of acetylcholine are stored in the nerves, with variable capacities to release upon stimulation. Collier and MacIntosh (1969) have suggested that the acetylcholine turn-over in the superior cervical ganglion in cats takes place in non-uniform pools and that the newly synthesized acetylcholine shows a preferential release upon preganglionic stimulation. An alternative explanation for the decline of the release observed in this study may be that a regulatory mechanism is present which after a time diminishes the release of acetylcholine. Besides 3H-acetylcholine, 3H-choline was also released into the incubation medium. As a rather long washing period (45 min) had been used before any fractions were collected it is probable that the 3H-choline was derived predominantly from intracellular sources. The tissue content of 3H-choline at the end of the experiment was rather low, however, which might indicate that 3H-choline was derived from some other kind of radioactive metabolite. It seems improbable that the 3H-acetylcholine released was responsible for the 3H-choline formation, since a rather high concentration of acetylcholine-esterase inhibitor was used and the release of the two metabolites showed a poor temporal correlation (Fig. 1). Other metabolites were also present in the washing medium and in the tissue. This study gives no definite information on the nature of UPF. It might represent such metabolites as phosphorylcholine, betaine or N,N-dimethylethanolamine.It has been suggested that 3Hphosphorylcholine and 3H-betaine may be synthesized from 3H-choline in a variety of tissues (Sung and Johnstone 1965, Collier and Lang 1969, Szerb 1975). In conclusion, it is proposed that the use of 3H-choline for labelling of intrinsic acetylcholine stores with tritium might be of value for studying acetylcholine release from the gland. Recently this approach was used to investigate the influence of P-adrenoceptor active drugs on the cholinergic transmission in the rabbit lacrimal gland (Aberg et al. 1978). I am indebted to Assistant Professor Rolf G. G . Andersson for valuable criticism of this work. Financial support was given by the Swedish Medical Research Council (878-04X-04498-04).
References ABERG,G.,
G. AOLERand J. WIKBERC,Inhibition and facilitation of lacrimal flow by beta-adrenergic drugs. Acra ophral. (Kbb.). In press. CLEMENT, J. G. and E. H. COLHOUN, Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ions. Canad. J . Physiol. Pharmncol. 1975. 53. 1089-1093. COLLIER, B. and C. LANC,The metabolism of choline by a sympathetic ganglion. Canad. J. Physiol. Pharmacol. 1969. 47. 119-121.
ACh SYNTHESIS IN RABBIT LACRIMAL GLAND
113
COLLIER, B. and F. C. MACINTOSH, The source for acetylcholine synthesis in a sympathetic ganglion. Canad. J. Physiol. Pharmacol. 1969. 47. 127-135. DEMTSCHENKO, J., Zur Innervation der Thranendruss. Pfliigers Arch. ges. Physiol. 1872. 7. 191-200. ICHIKAWA,A. and Y . NAKAJIMA, Electron microscope study o n the lacrimal gland of the rat. Jap. J . exp. Med. 1962. 77. 136-149. MIZUKAWA, T., T. OTORI,J. HARAand M. IGA, Histochemistry of the human lacrimal gland. Jap. J. Ophtalmol. 1962. 6. 17-23. PERT,C. B. and S. H. SNYDER,High affinity transport of choline into the myenteric plexus of guinea-pig intestine. J. Pharmacol. exp. Ther. 1974. 191. 102-108. SIMON.J. R., S. ATWEKand M. J. KUKOE,Sodium dependent high affinity choline uptake: A regulatory step in the synthesis of acetylcholine. J. Neurochemistry 1976. 26. 909-922. SUNG,C.-D. and R. M. JOHNSTONE, Evidence for active transport of choline in rat kidney cortex slices. Canad J. Biochemistry 1965. 43. 11 11-1 118. SZERB,J. C., Endogenous acetylcholine release and labelled acetylcholine formation from (3H)-choline in the myenteric plexus of the guinea pig ileum. Canad. J. Physiol. Pharmacol. 1975. 53. 566-574. TEPLIACHINE, A., Recherches sur les nerves secretoires de la glande lacrymale. Arch. opthal. (Paris) 1894. 14. 40 1-4 13. WALLACH, M. B., A. M. GOLDBERG and F. E. SHIDEMAN, The synthesis of labelled acetylcholine by the isolated cat heart and its release by vagal stimulation. I n t . J. Neuropharmac. 1967. 6. 317-323. WIKBERG, J., Release of 3H-acetylcholine from isolated guinea pig ileum. A radiochemical method for studying the release of the cholitiergic neurotransmitter in the intestine. Acta physiol. scand. 1977. 101. 302-3 17.
8 - 795871
Synthesis of 3H-acetylcholinein the rabbit lacrimal gland and its release by electrical field stimulation BY JARLE. S. WIKBERG
Received 17 May 1978
Abstract WIKBERG, J. E. S. Synthesis of 3H-acetylcholine in the rabbit lacrimalgland and its release by electrical field stimulation. Acta physiol. scand. 1979. 105. 108-1 13. Electrical field stimulation was applied to the rabbit lacrimal gland during incubation in uitro with 3Hcholine, after which the efflux of tritiated metabolites was studied. In the presence of physostigmine 3Hacetylcholine was released into the incubation medium when the gland was stimulated electrically. The identity of the 3H-acetylcholine synthesized by the preparation was established by its identical ion exchange chromatographic properties to native acetylcholine. The tissue contents of 3H-acetylcholine and 3H-choline were 14.4 and 3.6"/,, respectively, of the total activities found in the gland. This study demonstrates that the rabbit lacrimal gland is innervated cholinergically. It is suggested that the method developed might be of value for studying effects of drugs on cholinergic neurotransmission in this organ.
The main secretory innervation of the mammalian lacrimal gland is thought to be cholinergic (Demtschenko 1872, Tepliachine 1894, Arenson and Wilson 1971). In previous studies of the cholinergic neurotransmission in this organ measurement of tear secretion has generally been used for indirect determination of acetylcholine secretion from the nerves. Recently a radiochemical method for studying acetylcholine release from isolated guinea pig ileum was described (Wikberg 1977). In the present work a similar approach was used to study the release of acetylcholine from isolated rabbit lacrimal glands. Electrical field stimulation was applied to the organ during incubation with 3H-choline, whereafter the release of 3H-acetylcholine synthesized by the organ could be examined.
Methods Rabbits of either sex were killed by a blow on the neck and exsanguinated. One of the lacrimal glands was rapidly excised through the caudal supraorbital notch and its capsule was carefully dissected away with forceps. The organ (weighing 114k 11 mg), was mounted in a 4 ml water-jacketed perspex chamber containing Krebs solution (Wikberg 1977) with eserine sulphate 3 x M. The solution was kept at 37°C and gassed with a mixture of 0, (95 X) and CO, ( 5 %). The lacrimal gland was incubated with 9.3 x lo6 Bq (becquerel, dps) of 3H-choline (methyl 3H-choline chloride 3.7 x loL1Bq/mmole, Radiochemical Centre, Amersham, England) at a final concentration of 1 x lo-" M, for 1 h. During the incubation the organ was stimulated electrically by 0.1 ms, 320 mA rec-
108
ACH SYNTHESIS IN RABBIT LACRIMAL GLAND
--
Fig. 1. Release of tritiated metabolites from the lacrimal
1
109
Field stimulation 10Hz c
20-
S.E. of 5 expts.
Time (mid tangular pulses a t 0.1 Hz by means of two 2 0 1~mm platinum electrodes mounted 10 mm apart on each side of the organ, using a Grass SD9 stimulator and a home-constructed timer. After the incubation the electric stimulation was stopped and the organ was washed with Krebs solution at intervals of 5 min for 45 min, after which 4 ml fractions were collected for analysis. Rarliochemical assays The method described previously (Wikberg 1977), with some minor modifications, was used, to separate the radioactivity of the samples into 3 fractions: 3H-acetylcholine, ,H-choline and a fraction which remained unprecipitated by ammonium reineckate (UPF). In order to improve the paper chromatographic separation of acetylcholine and choline, a descending rather than the ascending system with acetone/water/lO N HCI 100:20:1 (by volume) was used. The chromatograms were allowed to run for 10 h, using the "durchlauf" technique. To facilitate the identification of carrier choline and acetylcholine the spots were developed by spraying with an iodoplatinate reagent (Walach et al. 1967). By this method the absolute distance between the spots was 3 cm. In order to elute the radioactivity from the paper and to decolorise the iodoplatinate reagent, a mixture of 3 ml ethanol/0.5 M NH, 1 : 1 (v/v) was used. These samples could be counted after addition of 1nstagelO (Packard Instrument Company Inc.) as described previously. After the expt. the lacrimal gland was frozen in Frigen solid CO, and kept at - 80°C until analysed. The tissue was homogenized with a glass homogenizer in 0.4 M HCIO, (1 0.5 ml) at 0°C and then centrifuged a t 10 000 x g for 30 min. The pellet was washed once with 1 ml of the HCIO, solution. Th e pooled supernatant was diluted to 25 ml and 1 ml fractions were taken for analysis as described above for the bathing solution. The pellet was dissolved by adding 2 ml Soluene 350@ (Packard Instrument Company Inc.) and counted in a Packard Tri Carb 3375 liquid scintillation spectrometer after addition of a toluenebased scintillator.
+
Ion exchange chromatography
An approach similar to that described previously was used (Wikberg 1977). Columns measuring 4.4 x 450 mm were packed with Amberlite C G 50 11 (200-400 mesh) equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. The samples applied on the column were eluted with 5 ml H,O followed by the phosphate buffer a t a flow rate of 5 ml/h. In order to determinate the retention volume of acetylcholine, 1.3 x lo3 Bq of 14C-acetylcholine (A~etyI-l-~~C-acetylcholine iodide 8.5 x I O'O Bq/mmole, NEN) was added to the sample and the radioactivity was separated from the tritium activity by measurement at separate channels in the
110
JARL E. S. WIKBERG
I
!'
Fig. 2. Ion exchange chromatography of the reineckate-precipitated fraction in the washing solution from the lacrimal gland. The medium was collected during electrical field stimulation a t 10 Hz and the quaternary ammonium compounds were precipitated by addition of ammonium-reineckate in the presence of carrier acetylcholine and choline. After batch treatment with Biorex 9 in water and addition of 1.3 2 lo3 Bq, 14C-acetylcholine as a marker, the sample was applied on the column, followed by elution with 5 ml water and 0.1 M phosphate buffer, pH 7.0. Insert: Correlation between the 3H-a~tivityin the first peak and the 14C-activity of the 14C-acetylcholine marker (r = 1.000).
liquid scintillation spectrometer. The data obtained were corrected for quenching and for overlap between channels, using an internal standard and a double isotope computer programme written for HP9830. The counting efficiency was 15 "b for 3H and 53 "b for 14C.
Results During unstimulated conditions almost no tritiated acetylcholine was released from the organ. After the 45-min washing period when collection of fractions was started, only 2% of the radioactivity released to the bathing solution consisted of 3H-acetylcholine. Most of the activity at this time consisted of UPF (79%), followed by 3H-choline (19%). Electrical field stimulation with 1.0 ms pulses at 10 Hz induced a 21-fold increase in the release of 3Hacetylcholine. The release of acetylcholine was not maintained at that level but decreased to half its maximal value in approximately 10 min, despite continuous electrical stimulation. The electrical stimulation also induced a two-fold increase in the release of 3H-choline, and during the subsequent stimulation periods this release showed a smaller tendency to decline than that of 3H-acetylcholine. The release of UPF also increased approximately 2-fold on electrical stimulation (Fig. I). The identity of 3H-acetylcholine released during electrical stimulation was verified by ion exchange chromatography. After application of the reineckate precipitated fraction on the column two peaks of radioactivity appeared. The first peak showed excellent correlation to the marker 14C-acetylcholinepeak (correlation coefficient 1 .OOO) (Fig. 2).
ACh
SYNTHESIS IN RABBIT LACRIMAL G L A N D
111
TABLEI. Tritiated metabolites present in the lacrimal gland at the end of the expt. The radioactivity was separated into 4 fractions: the ammonium reineckate unprecipitated fraction (UPF), acetylcholine (Ach), choline (Chol) and the perchloric acid insoluble fraction (Pellet). The table gives the mean S.E. of 5 expts. x Bq
UPF
Ach Chol Pellet
* *
7.55 1.02 2.85k0.35 0.72 & 0.07 8.68 1.44
% 38.1
14.4 3.6
43.8
When the expt. started the tissue content of tritium was 2.18 10.21 y lo5 Bq. This amount corresponded to 23.4% of the total activity of 3H-choline added to the incubation medium at the start of the expt. During the course of the expt. a total of 1.99 k0.60 x lo4 Bq of tritium was released from the lacrimal gland. The secretion of 3H-acetylcholine was 1.22 k 0.10 x lo3 Bq, of 3H-choline 2.93 k0.30 x lo3 Bq and of UPF 1.64 rtr0.77 x lo4 Bq. At the end of the expt. a considerable amount of 3H-acetylcholine was present in the tissue but the 3H-choline content was low. The largest amounts of activity, however, were found as UPF and in the PCA-insoluble fraction (Table I).
Discussion The innervation of the mammalian lacrimal gland has been studied by histological techniques in several earlier studies. Ichikawa and Nakajima (1962) showed that the lacrimal gland of the rat contains unmyelinated nerve fibres in the inter- and intra-lobular connective tissues. Electron microscopic studies on lacrimal glands of monkeys have indicated that parasympathetic nerve fibres originating in the sphenopalatine ganglion innervate interstitial and parenchymal tissues (Ruskell 1969). The arteriole of the monkey lacrimal gland may receive both cholinergic and sympathetic nerves (Ruskel 1967, 1969). Histochemical staining for cholinesterase in the human lacrimal gland has revealed choline esterase activity in myoepithelial cells and nerve fibres surrounding the acinar cells (Mizukawa ef al. 1962). The present study demonstrates that the rabbit lacrimal gland is capable of accumulating tritium when incubated with 3H-choline. It may be estimated that a passive uptake of choline of about 5 % of the amount in the incubation medium will take place under the experimental conditions used. The high uptake observed (23.4 % of the given amount) therefore suggests that an active mechanism for choline transport into the tissue is present. This transport system could be located in nervous tissue as well as in glandular cells. Active uptake of choline into nervous tissue is well documented (Pert and Snyder 1974, Simon et al. 1976, Wikberg 1977), but other cells such as human red blood corpuscles may have transport systems for choline accumulation (Clement and Colhoun 1975). In this work it was found that the rabbit lacrimal gland is capable of synthesizing 3Hacetylcholine from 3H-choline. The chemical identity of 3H-acetylcholine seems to be certain, since the metabolite showed properties identical to those of native acetylcholine in two different chromatographic systems. The basal release of 3H-acetylcholine into the in-
I12
JARL E. S. WIKBERC
cubation medium was almost negligible. This contrasted markedly with the results obtained from guinea pig ileum (Wikberg 1977), where the unstimulated release was much higher and showed spontaneous fluctuations. Electrical stimulation, with stimulation parameters indicating that selective stimulation of nervous tissue was induced, resulted in a large increase of the release of 3H-acetylcholine from the lacrimal gland. The results of these experiments may be taken as fairly clear evidence of the presence of cholinergic innervation in this tissue. The tissue content of 3H-acetylcholine was more than 20-fold larger than the amount of 3H-acetylcholine released during the electrical stimulation. In spite of this the release of 3Hacetylcholine declined rapidly after the initial large release. This observation might indicate that different compartments of acetylcholine are stored in the nerves, with variable capacities to release upon stimulation. Collier and MacIntosh (1969) have suggested that the acetylcholine turn-over in the superior cervical ganglion in cats takes place in non-uniform pools and that the newly synthesized acetylcholine shows a preferential release upon preganglionic stimulation. An alternative explanation for the decline of the release observed in this study may be that a regulatory mechanism is present which after a time diminishes the release of acetylcholine. Besides 3H-acetylcholine, 3H-choline was also released into the incubation medium. As a rather long washing period (45 min) had been used before any fractions were collected it is probable that the 3H-choline was derived predominantly from intracellular sources. The tissue content of 3H-choline at the end of the experiment was rather low, however, which might indicate that 3H-choline was derived from some other kind of radioactive metabolite. It seems improbable that the 3H-acetylcholine released was responsible for the 3H-choline formation, since a rather high concentration of acetylcholine-esterase inhibitor was used and the release of the two metabolites showed a poor temporal correlation (Fig. 1). Other metabolites were also present in the washing medium and in the tissue. This study gives no definite information on the nature of UPF. It might represent such metabolites as phosphorylcholine, betaine or N,N-dimethylethanolamine.It has been suggested that 3Hphosphorylcholine and 3H-betaine may be synthesized from 3H-choline in a variety of tissues (Sung and Johnstone 1965, Collier and Lang 1969, Szerb 1975). In conclusion, it is proposed that the use of 3H-choline for labelling of intrinsic acetylcholine stores with tritium might be of value for studying acetylcholine release from the gland. Recently this approach was used to investigate the influence of P-adrenoceptor active drugs on the cholinergic transmission in the rabbit lacrimal gland (Aberg et al. 1978). I am indebted to Assistant Professor Rolf G. G . Andersson for valuable criticism of this work. Financial support was given by the Swedish Medical Research Council (878-04X-04498-04).
References ABERG,G.,
G. AOLERand J. WIKBERC,Inhibition and facilitation of lacrimal flow by beta-adrenergic drugs. Acra ophral. (Kbb.). In press. CLEMENT, J. G. and E. H. COLHOUN, Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ions. Canad. J . Physiol. Pharmncol. 1975. 53. 1089-1093. COLLIER, B. and C. LANC,The metabolism of choline by a sympathetic ganglion. Canad. J. Physiol. Pharmacol. 1969. 47. 119-121.
ACh SYNTHESIS IN RABBIT LACRIMAL GLAND
113
COLLIER, B. and F. C. MACINTOSH, The source for acetylcholine synthesis in a sympathetic ganglion. Canad. J. Physiol. Pharmacol. 1969. 47. 127-135. DEMTSCHENKO, J., Zur Innervation der Thranendruss. Pfliigers Arch. ges. Physiol. 1872. 7. 191-200. ICHIKAWA,A. and Y . NAKAJIMA, Electron microscope study o n the lacrimal gland of the rat. Jap. J . exp. Med. 1962. 77. 136-149. MIZUKAWA, T., T. OTORI,J. HARAand M. IGA, Histochemistry of the human lacrimal gland. Jap. J. Ophtalmol. 1962. 6. 17-23. PERT,C. B. and S. H. SNYDER,High affinity transport of choline into the myenteric plexus of guinea-pig intestine. J. Pharmacol. exp. Ther. 1974. 191. 102-108. SIMON.J. R., S. ATWEKand M. J. KUKOE,Sodium dependent high affinity choline uptake: A regulatory step in the synthesis of acetylcholine. J. Neurochemistry 1976. 26. 909-922. SUNG,C.-D. and R. M. JOHNSTONE, Evidence for active transport of choline in rat kidney cortex slices. Canad J. Biochemistry 1965. 43. 11 11-1 118. SZERB,J. C., Endogenous acetylcholine release and labelled acetylcholine formation from (3H)-choline in the myenteric plexus of the guinea pig ileum. Canad. J. Physiol. Pharmacol. 1975. 53. 566-574. TEPLIACHINE, A., Recherches sur les nerves secretoires de la glande lacrymale. Arch. opthal. (Paris) 1894. 14. 40 1-4 13. WALLACH, M. B., A. M. GOLDBERG and F. E. SHIDEMAN, The synthesis of labelled acetylcholine by the isolated cat heart and its release by vagal stimulation. I n t . J. Neuropharmac. 1967. 6. 317-323. WIKBERG, J., Release of 3H-acetylcholine from isolated guinea pig ileum. A radiochemical method for studying the release of the cholitiergic neurotransmitter in the intestine. Acta physiol. scand. 1977. 101. 302-3 17.
8 - 795871
