[ 143 ] DISTRIBUTION OF ACETYLCHOLINE AND HISTAMINE IN NETTLE PLANTS BY N. EMMELIN* (LUND) AND W. EELDBERGf From the Physiological Laboratory., University of Cambridge In a previous paper we have described the occurrence of acetylcholine, histamine and a third unidentified smooth muscle-contracting substance in the hair fluid of Urtica urens, the annual nettle. The vascular reactions produced in the human skin and the sensations elicited by the sting of the nettle hair could be accounted for by the combined effect of acetylcholine and histamine. Both substances were also found in the leaf tissue (Emmelin & Feldberg, 1947). In the experiments described in the present paper it could be shown that acetylcholine and histamine occur also in high concentrations in the hair fluid of the perennial nettle, U. dioica. No experiments have been carried out on the occurrence of the third unidentified smooth muscle-contracting substance, but the distribution of histamine and acetylcholine in the annual and perennial plant have been examined in more detail. Since the publication of our previous experiments our attention has been drawn to a paper by Starkenstein & Wasserstrom (1933) who had found that extracts of whole nettle plants caused contractions of smooth muscles, a fall in arterial blood pressure and inhibition of the heart. No indication was given as to the nature of the substance or substances responsible for these effects. In the light of the experiments presented in this paper, histamine and acetylcholine were probably the active principles, or at least the main active principles in their extracts. METHODS The procedure adopted was the same as that described previously. For the routine assay of acetylcholine the eserinized frog rectus muscle was used. For the identification the following tests were applied: sensitivity to alkali and to cholinesterase, stability to boiling in acid solution, the stimulating eflect on the isolated guinea-pig's intestine, the abolition of the effect by atropine but its resistance to benadryl. For the routine assay of histamine the atropinized guinea-pig's intestine preparation was used; for the identification use was made of the antagonistic action of neoantergan. All extracts were made with saline or acidified saline. The completeness of the extraction was controlled by showing that re-extraction of the insoluble material with acid saline and boiling yielded no active substance or traces only of histamine and acetylcholine. The values for histamine refer to the dihydrochloride, those for acetylcholine to the chloride. RESULTS Hairs. The fluid from the hairs of U. dioica contains acetylcholine as well as histamine (Table i). The acetylcholine content per individual hair was usually between 0-04 and 0-07 y, but in one experiment with particularly small hairs the value was 0-013 7- There * With a grant from the University of Lund Fund for the Advancement of Medical Research, t With a grant from the Medical Research Council.
144 N. EMMELIN AND W. FELDBERG was no difference between the content of acetylcholine in the hairs from the main stems and from the leaves. In corresponding experiments with JJ. iirens the hairs from the stem had been found usually to contain a larger amount of acetylcholine than those of the leaves. This difference was attributed to the larger size of the hairs situated on the stems. In U. dioica there is no obvious difference in the size of the hairs situated on the stems or on the leaves. The histamine content was greater in the hairs from the stem than from the leaves. Similar results had been obtained in the experiments with U. urens. Table i. Acetylcholine and histamine content of hairs from Urtica dioica Origin of hairs No, of hairs used for assay Acetylcholine in y per hair Histamine in y per hair Stem Leaves Stem Leaves Stem (small hairs) 30 30 SOO 500 100 7 0 046 0-040 0-049 0-013 o-oi 0-0035 Leaves. In order to obtain correct values it is necessary to remove as carefully as possible the hairs before extracting the leaf tissue, otherwise the values obtained are too high. For instance, two samples of leaf tissue from adjacent leaves of U. dioica were prepared, care being taken that the larger veins of the leaves were not included in the samples. In the one sample the hairs were left on the leaf tissue and included in the extraction; the acetylcholine content was 0-7 y/io mg. tissue. In the other sample, in which the hairs had been removed before extraction, the value was 0-095 y/io mg. tissue only. The corresponding values for histamine in the two samples were 0-3 and y respectively. As long as the hairs remained on the leaf tissue the values obtained for acetylcholine were higher than those for histamine, but when the hairs had been removed the order was more often reversed. It is possible that incomplete removal of the hairs was the reason why this reversal did not always happen. The histamine and acetylcholine extracted from leaf tissue is not derived, or derived specially, from those small spots from which the hairs originate, i,e. the bases of the hairs, because the results were the same if the small areas of tissues used for extraction were carefully cut out between the hairs. The results for both U. urens and dioica are given in Table 2. When both acetylcholine Table 2. Acetylcholine and histamine in y per 10 mg. leaf tissue [hairs removed) U. urens: Acetylcholine Histamine U. dioica: Acetylcholine Histamine 0-09 10 0-095 0-2 0-14 10 0-095 oos 0-83 0-67 0-2 002 0-005 0-27 0-35 3 0-18 0-19 0-12 0-5 0-21 0-3 0-65 and histamine were assayed in the same sample the two values are given below each other in the table. It will be seen that there are great individual variations, but that apart from these the histamine concentration is higher in the leaf tissue of U. urens than of U. dioica. Stems. When the hairs had been removed carefully the values for acetylcholine varied between less than o-i and more than i-oy/iomg. tissue; those for histamine between 0-017 '^^^ o-7y/iomg. tissue. The two substances were not found to be uniformly
Distribution of acetylcholine and histamine in nettle plants 145 distributed in the stem tissue. When the greenish or greenish reddish chlorophyllcontaining outer layer, the assimilating cortex, was peeled oflf and examined separately from the stele, it was found to contain both substances in higher concentration (Table 3). The concentration of acetylcholine in the assimilating cortex is on the average higher than that found in the leaf tissue. The increase is particularly pronounced in U. urens. The histamine concentration in the assimilating cortex of the stems of U. dioica, on the other hand, is certainly lower than that of the leaf tissue, whereas in U. urens the concentration in these two tissues is practically the same. Roots and rhizomes. No histamine could be detected in any part of the roots and rhizomes of U. dioica, whereas it occurred in relatively high concentrations in the roots of U. urens. Acetylcholine was present in the roots of both plants, but in much lower concentration in U. urens than in U. dioica. Table 3. Distribution of acetylcholine and histamine in outer and inner layer of stem Content in y per io mg. tissue Plant Assimilating cortex Acetylcholine Stele Assimilating cortex Histamine Stele U. iirens U. urens U. dioica U. dioica U. dioica 0-45 o-s o'26 0 07 o-s O'o6 on O-O2 O'i4 II O-8 0-03 0-05 0-23 0-013 o-oi Table 4. Distribution of acetylcholine and histamine in the underground tissue of Urtica urens Exp. no. Acetylcholine in y per lo mg. tissue Main root Rootlets Whole Cortex Stele Histamine in y per io mg. tissue Main root Rootlets Cortex Stele I 2 3 4 S 6 8 Mean 0-55 0-19 0-14 0-13 0-13 0-23 0-08 0-03 0-09 0-05 0-24 0-08 003 0-54 o-oi 0-02 O-OI 0-025 i-i 055 0-8 0-38 0-7 2-7 i-o 0-65 0-36 In Table 4 is shown the distribution of acetylcholine and histamine in the underground tissue of U. urens. The values vary greatly in different plants, but the differences between the different parts of the roots are evident in all experiments. The mean values are given at the bottom of the table. The small rootlets contain acetylcholine in a higher concentration than the main roots. In these the concentration of acetylcholine is highest in the cortex, but even here not as high as in the rootlets. Histamine also is more concentrated in the cortex than in the central stele of the main roots, but there is no constant difference between rootlets and main roots. In all parts the concentration of histamine was found
146 N. EMMELIN AND W, FELDBERG to be greater than that of acetylcholine. The histamine concentration of the rootlets and of the cortex of the main root was of the same order as that observed in leaf tissue and in the assimilating cortex of the stems. No histamine could be found in any part of the underground tissue of U. dioica, although the sensitivity of the assay was such that amounts of histamine greater than 0-005 y/i n^g- tissue would have been detected. The acetylcholine was present in the rootlets, roots and rhizomes. The rootlets contained between 0-45 and 1-97 acetylcholine in 10 mg. tissue, usually the finest hairlets giving the lowest values. The rhizomes contained, on the other hand, as much as 3-7 y of acetylcholine in 10 mg. tissue. The greatest proportion of this acetylcholine is derived from the brownish, cortex and the adjacent greenish phloem. In these between 5-6 and 135 y (average 37 y) per 10 mg. tissue were found, an average concentration of acetylcholine of about i : 250, and in one instance even of about i : 70. Synthesis of acetylcholine. In a few experiments the methods described by Feldberg (1945) and by Feldberg & Mann (1945) for the synthesis of acetylcholine in animal tissue were applied to saline extracts prepared either from fresh or from acetone-dried tissue of the leaves, roots and rhizomes of the nettle plant. No synthesis occurred on incubation of these extracts for 2-3 hr. at 25 or 37 C. under aerobic or anaerobic conditions. Table 5. Distribution of acetylcholine in different parts of rhizomes of Urtica dioica Exp. no. Outer part (whole) Cortex Phloem Inner part Wood Pith I 5-6 2 16 3 i 4 22 28 5 13s O'4 o-i 6 24 24 2-9 II 7 25 4 O-9 8 54 2-6 10 DISCUSSION Our results show that acetylcholine as well as histamine, which previously had been found in the hair fluid of U. tirens, occur also in the hair fluid of U. dioica, the perennial plant. This was to be expected, since these substances were found to be responsible or at least mainly responsible for the reactions of the human skin to the nettle sting. Both substances were found to occur not only in the hair fluid, but also in other parts of the plant. A study of their distribution revealed differences between U. urens and dioica. In U. urens the concentrations of acetylcholine and of histamine appears to be of the same order in the leaves, in the outer cortex of both stem and main root, and in the rootlets. At least the variations between individual plants and between individual values obtained from the same part of different plants are so great as to render it impossible to detect, with the relatively few determinations so far made, for each tissue, significant differences. The concentration of acetylcholine in these tissues varied between i in 500,000 and I in 20,000 with a mean concentration of i in 50,000. The mean concentration for the separate tissues was: leaf i in 80,000; assimilating cortex of stem i in 20,000; cortex of main root i in 60,000 and rootlets i in 40,000. The concentration of histamine in these tissues varied from about i in 50,000 to i in 4000 with a mean concentration of i in 10,000.
Distribution of acetylcholine and histamine in nettle plants 147 The mean concentration for the separate tissues was: leaf i in 14,000; assimilating cortex of stem I in 10,000; cortex of main root i in 8000 and rootlets i in 12,000. These concentrations are much weaker than those observed in the hair fluid. The substances may therefore be transported to the hairs and there concentrated. In that case acetylcholine would have to be concentrated to a greater extent than histamine. In U. dioica acetylcholine and histamine are not uniformly distributed. Despite the great individual variations found it could be shown that there are real differences in the various tissues. The mean concentration of acetylcholine in the leaves and in the assimilating cortex of the stem, i.e. the green tissues, was about equal, over i in 40,000, but increased to i in 9000 in the rootlets, to i in 2000 in the rhizome and in its cortex and phloem to even i in 250. Histamine was not found in all parts of the plant; it was absent in the rhizomes, roots and rootlets. Its concentration in the leaves was about four times that in the assimilating cortex of the stems where it occurred in a mean concentration of I in 250,000 only. It was hoped to obtain from the distribution of acetylcholine and histamine in the different parts of the nettle plants some information about the possible site of the formation of these substances. The rather uniform distribution, below and above ground, in the soft tissues of U. urens made it impossible to obtain any indication in which special tissue these substances might be formed in the annual plant. But also the uneven distribution of these substances in the perennial plant does not allow us to state with certainty the tissue responsible for their formation. It may well be that the very high concentration of acetylcholine in the rootlets and rhizomes is not associated with its formation here, but with the fact that it has accumulated in these perennial parts during previous years. If the acetylcholine were formed below the ground it might be formed either by soil bacteria or by a tissue enzyme present in the rootlets and in the cortex and phloem of the rhizome; if formed by the action of a tissue enzyme the conditions for its activity, however, would be different from those which favour the synthesis of acetylcholine in animal tissue. From the distribution of histamine in the different parts of U. dioica, outside the hair ffuid the most likely tissue for the site of its formation is the green tissue of the leaves, where it might be formed either by a tissue enzyme or even by ultra-violet decarboxylation of histidine. SUMMARY 1. The presence of acetylcholine and histamine in high concentrations in the fluid of the hairs, previously found for Urtica urens has now also been established for U. dioica, the perennial plant. 2. The distribution of acetylcholine and histamine in the different tissues of the annual and perennial nettle plant has been examined. 3. In U. urens acetylcholine and histamine are rather uniformly distributed in the following parts: the leaves, the outer cortex of both stem and main root, and the rootlets. The concentration in these tissues varies greatly in different plants, the mean concentration is I in 50,000 for acetylcholine and i in 10,000 for histamme. 4. In U. dioica acetylcholine and histamine are not so uniformly distributed as in U. urens. The mean concentration for acetylcholine in the leaf and the assimilating cortex of the stem was over i in 40,000, in the rootlets i in 9000, in the rhizome i in 2000 and in its cortex and phloem even i in 250. Histamine was not present in the underground
148 N. EMMELIN AND W. FELDBERG tissue. It occurred in the leaves in a mean concentration of about i in 60,000 and in the assimilating cortex of the stem of about i in 250,000. 5. Acetylcholine and histamine may not be formed in the hairs but transported to them and there concentrated. The possible site and mode of formation of these substances in nettle plants is discussed. We should like to make grateful acknowledgement to Dr K. R. Sporne of the Botany School, Cambridge, for aiding us in the anatomy of the plants. REFERENCES EMMELIN, N. & FELDBERG, W. (1947). J. Physiol. 106, 440. FELDBERG, W. (1945). J. Physiol. 103, 367. FELDBERG, W. & MANN, T. (1945). J. Physiol. 104, 411. STARKENSTEIN, E. & WASSERSTROM, T. (1933). Arch. exp. Path. Pharmak. 172, 137. [Received 6 April 1948)