Quantitation of peptide hormone in single cultured secretory neurons of the crab,Cardisoma carnifex

The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1–50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of 150 peptidergic neurons that form the X-organ—sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having high (>33 pg; mean 86±5, S.E., n=47) or low (33pg; mean 22±2.5; n=17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2±2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.     

Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Ultrastructural changes in isolated peptidergic nerve terminals induced by digitonin permeabilization and K+ stimulation in the sinus gland of the crab,Cardisoma carnifex

Summary The preparation of isolated peptidergic nerve terminals from the sinus gland (a neurohemal organ) of the crab (Cardisoma carnifex) is described. In this species the nerve endings can have diameters up to 30 m. They release neurosecretory material as judged by the decrease in the volumetric density of granules upon depolarization with potassium. Similar results were obtained after permeabilization of the nerve terminals with digitonin, but only in the presence of micromolar concentrations of calcium. This preparation should prove useful in correlating electrical events with other cellular processes involved in stimulus-secretion coupling.     

Immunochemical evidence for the transport of neurophysin in the hypothalamo-neurohypophysial system of the dog

Summary 1. An immunohistochemical study has been made of the hypothalamo-neurohypophyseal system of the dog, 20h after crushing the pituitary stalk.2. By use of a cross-species-reactive neurophysin antiserum it was shown that neurophysin is a component of the axons which originate in the supraoptic and paraventricular nuclei and terminate around blood vessels in the posterior pituitary.3. Neurophysin specific fluorescence accumulated in axons proximal to the constriction but was absent from the axons immediately distal to the site of injury.4. In dogs left for six days it was shown by radioimmunoassay that the amount of neurophysin in the hypothalamus and stalk proximal to the constriction increased twofold while that remaining in the posterior pituitary and stalk distal to the constriction decreased five-fold over the same period.5. The results are interpreted as evidence for a rapid axonal transport of neurophysin from its site of synthesis in the cell bodies of the hypothalamus to the posterior pituitary. Acknowledgements: This work was supported by a research grant to D. B. Hope from the Medical Research Council. L. O. Uttenthal was supported by a Medical Research Training Award and B. G. Livett by a Nuffield Dominions Trust Demonstratorship (Australia). We thank Mrs. Marion Martin for radioimmunoassay of neurophysin, Miss Wendy Jones for technical assistance and the U.C.L.A. Brain Information Service for help with the bibliography.     

Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin

Translocation of [¹⁴C]gibberellic acid into antheridial cells of Chara vulgaris L. was investigated in relation to the presence of symplasmic connections between the antheridium and the thallus. It was found that manubria, capitular cells, and antheridial filaments were about three-fold more strongly labelled in young antheridia connected to the thallus by plasmodesmata than in older antheridia in which spontaneous symplasmic isolation had occurred. Plasmolytically induced symplasmic isolation of young antheridia severely diminished the radioactivity of all the cells, down to the level characteristic for spontaneously isolated antheridia. It is concluded that plasmodesmata are the main channel of gibberellin transport into antheridia. The change in the character of symplasmic connections during the course of morphogenesis might, among other events, constitute a signal determining a shift of cell metabolism in a new direction, in response to a rapid change in gibberellin level.Abbreviations GA_(n) gibberellin (A_n) - GA₃ gibberellic acid - IAA indole-3-acetic acid This study was supported by the Polish Academy of Sciences research project CPBP 04.01.5.05.     

The organization of serotonin-immunoreactive neuronal systems in the brain of the crested newt,Triturus cristatus carnifex Laur.

Summary The distribution of serotonin (5-HT) immunoreactive structures has been investigated in the brain of the crested newt by means of indirect immunofluorescence, and unlabeled antibody peroxidase-antiperoxidase-complex (PAP) or biotin-avidin-system (BAS) techniques. In the newt, the bulk of the serotoninergic system extends from the raphe region of the medulla oblongata, through the isthmus, toward the mesencephalic tegmentum, and is characterized by pyriform neurons mainly located in a subependymal position, close to the midline. Also in the caudal hypothalamus, in addition to some 5-HT-positive adenohypophysial cells, many immunoreactive CSF-contacting neurons are found lining the paraventricular organ and the nucleus infundibularis dorsalis. A rich serotoninergic innervation was observed in the preoptic area and in the habenular complex. Concerning the telencephalon, immunopositive nerve fibers are encountered in the dorsal pallium, primordium hippocampi, striatum and olfactory bulbs. The general organization of serotoninergic systems in the newt brain exhibit close similarities to that described in higher vertebrates.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

Fine structure of the neurohemal sinus gland of the shore crab,Carcinus maenas,and immuno-electron-microscopic identification of neurosecretory endings according to their neuropeptide contents

Summary The sinus gland of the shore crab, Carcinus maenas, is a compact assembly of interdigitating neurosecretory axon endings abutting upon the thin basal lamina of a central hemolymph lacuna. Four types of axon endings are distinguishable by the size distribution, shape, electron density and core structure of their neurosecretory granules. One additional type of axon ending is characterized by electron-lucent vacuoles and vesicles. The axon profiles are surrounded by astrocyte-like glial cells. Various fixations followed by epoxy- or Lowicrylembedding were compared in order to optimize the preservation of the fine structure of the granule types and the antigenicity of their peptide hormone contents. By use of specific rabbit antisera, the crustacean hyperglycemic, molt-inhibiting, pigment-dispersing, and red-pigment-concentrating hormones were assigned to the four distinct granule types which showed no overlap of immunostaining. Epi-polarization microscopy and ultrathin section analysis of immunogold-stained Lowicrylembedded specimens revealed that immunoreactivity to Leu-enkephalin and proctolin is co-localized with moltinhibiting hormone immunoreactivity in the same type of granule. The size and core structure of the immunocytochemically identified granule types vary little with the different pretreatments but, in some cases, to a statistically significant extent. The present results are compared with those from earlier studies of sinus glands in different crustaceans. The methods of granule identification used in this study supplement the classical approach in granule typing; they are easier to perform and more reliable for the analysis of release phenomena in identified secretory neurons supplying the neurohemal sinus gland.     

Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants

The movement of systemin, the 18-amino-acid polypeptide inducer of proteinase inhibitors in tomato (Lycopersicon esculentum L.) plants, was investigated in young tomato plants following the application of [¹⁴C]systemin to wounds on the surface of leaves. Wholeleaf autoradiographic analyses revealed that [¹⁴C]systemin was distributed throughout the wounded leaf within 30 min, and then during the next several hours was transported to the petiole, to the main stem, and to the upper leaves. The movement of [¹⁴C]systemin was similar to the movement of [¹⁴C]sucrose when applied to leaf wounds, except that sucrose was slightly more mobile than systemin. Analyses of the radioactivity in the petiole phloem exudates at intervals over a 5-h period following the application of [¹⁴C]systemin to a wound demonstrated that intact [¹⁴C]systemin was present in the phloem over the entire time, indicating that the polypeptide was either stable for long periods in the phloem or was being continually loaded into the phloem from the source leaf. The translocation pathway of systemin was also investigated at the cellular level, using light microscopy and autoradiography. Within 15 min after application of [³H]systemin to a wound on a terminal leaflet, it was found distributed throughout the wounded leaf and was primarily concentrated in the xylem and phloem tissues within the leaf veins. After 30 min, the radioactivity was found mainly associated with vascular strands of phloem tissue in the petiole and, at 90 min, label was found in the phloem of the main stem. Altogether, these and previous results support a role for systemin as a systemic wound signal in tomato plants.The authors acknowledge the Washington State University Electron Microscope Center and staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Dr. Steven Doares for providing [³H]systemin. This research was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants IBN 9117795 and IBN 9104542     

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-³H]1-N-naphthylphthalamic acid and 3-indolyl-[2-¹⁴C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (K_D at 10^-8mol·l^-1) for one site and low affinity (K_D at 10^-6 mol·l^-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (K_D below 10^-8 mol·l^-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.Abbreviations IAA Indol-3-yl acetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid W.M. was supported in part by an allowance from CSIRO and in part by a fellowship of the Deutsche Forschungsgemeinschaft; he acknowledges the friendly hospitality of the CSIRO Division of Plant Industry. The authors thank R. Hertel (Freiburg) for valuable discussion.     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

The distribution of an immunoreactive endothelin-like substance in the nervous system of the nereid,Neanthes diversicolor (Annelida)

Summary The distribution of an immunoreactive endothelin-1-like peptide was investigated in the nereid, Neanthes diversicolor, using an antiserum raised against synthetic endothelin-1. Immunoreactive perikarya were localized in the brain, and nerve fibers containing endothelin-1-like material were found in the neuropil occupying the central portion of the brain. No immunostained fiber elements were traced in the circumesophageal connectives. Immunoreactive perikarya occurred in the subesophageal ganglion. From this ganglion, specifically stained fibers run posteriorly toward the ventral nerve cord. In each segmental ganglion, immunoreactive neurons were observed in medio-ventral and latero-ventral regions, and one or two marked fibers extended to the parapodium. In the parapodium, small immunoreactive perikarya and fiber elements were visible. Immunolabeled fibers occurred in the stomatogastric nerves, in the wall of the buccal cavity, and in the pharynx, esophagus, intestine and its anal region. Immunoreactive perikarya and nerve fibers were visualized between the circular muscle layer and epithelial cell layer in the esophagus and intestine. The endothelin-1-like substance shown to occur in N. diversicolor appears to function as a neurotransmitter or neuromodulator.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Immunohistochemical evidence for the existence of novel mammalian neuropeptides related to the Hydra GLW-amide neuropeptide family

The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH₂. To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons. This work was supported by grants from the Ministry of Education, Sports, and Culture, Japan.     

The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday     

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study

Neuroanatomic comparison of the binding capability of 2-[¹²⁵I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[¹²⁵I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.     

Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus

Summary The distribution of substance P-like immunoreactivity in the stomatogastric nervous systems of three decapod crustacean species, Cancer borealis, Homarus americanus, and Panulirus interruptus, was studied. The stomatogastric ganglion showed dense staining in the neuropil, but none in the somata. A single neuron stained in the esophageal ganglion. Lucifer yellow backfills and intracellular injections followed by incubation with the substance P antibody showed that the axons of this neuron project into the inferior esophageal nerves towards the paired commissural ganglia. The commissural ganglia showed a pronounced projection from a large bundle of fibers in the anterior medial portion of the circumesophageal connective. Additionally, less dense neuropil and stained somata were seen in the commissural ganglia. Staining was completely blocked by preabsorption with authentic substance P, physalaemin, eledoisin, and substance K. These data suggest that in the nervous system of crustacean species a molecule with C-terminal homology to substance P and other tachykinins is released as a neuroregulator in the stomatogastric ganglion.     

Autoradiographic demonstration of specific binding sites for E. coli enterotoxin in various epithelia of the North American opossum

Summary In the North American opossum, heat-stable specific binding sites for E. coli enterotoxin are observed (i) in epithelial cells lining the small intestine, colon, gall bladder, cystic duct, common bile duct and trachea, and (ii) in epithelial cells forming the duodenal (Brunner's) glands, liver, kidneys (metanephros, mesonephros) and testis, as demonstrated by autoradiography. Enterotoxin-specific binding sites in the intestinal tract are only found in intestinal epithelial cells with the highest concentration in the microvillus border. Enterotoxin-specific binding sites also occur in epithelial cells comprising the secretory tubules and ducts of the duodenal glands. In the kidneys (metanephros and mesonephros), enterotoxin-specific binding sites are confirmed primarily to the proximal tubules, whereas in the testis they are localized in seminiferous tubules. In the liver, enterotoxin-specific binding sites are confined primarily to hepatocytes. E. coli enterotoxin caused a 7-fold increase of cGMP in the liver and a 30-fold increase in the duodenal glands. The liver responded in about half of the animals studied, whereas the duodenal glands gave a consistent response in each case. Likewise, the duodenal glands consistently showed strong labelling for ¹²⁵I-enterotoxin, whereas receptor labelling of hepatocytes was inconsistent in nearly half the incubations and corresponds to the observed cGMP measurements.Supported by a Weldon Springs Grant, University of Missouri and by funds from the Medical Research Service, Department of Veteran's Affairs