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The effects of Na butyrate on the differentiation of newborn rat sympathetic neurons in primary cultures have been studied. Butyrate did not affect the long-term survival of these neurons in the presence of optimal concentrations of nerve growth factor, but decreased in a dose-dependent manner their protein content. In the range, 0.5-20 mM, butyrate did not modify the specific activity of lactate dehydrogenase in these cultures. Choline acetyltransferase activity developed at a 4.5- to 12-fold higher rate in cultures grown with 1-5 mM butyrate than in its absence. Concomitantly, tyrosine hydroxylase, dopa decarboxylase, dopamine-beta-hydroxylase, and acetylcholinesterase were depressed in cultures grown with butyrate. The deficit in acetylcholinesterase total activity was accompanied by an inhibition of the development of the asymmetric 16 S form of the enzyme. The deficit in tyrosine hydroxylase activity did not result from either a modification of the app Km for the enzyme's cofactor or a modification of its state of cAMP-dependent phosphorylation, but from a decrease in the number of immunoprecipitable enzyme molecules. A similar result was obtained with acetylcholinesterase. Butyrate thus reproduced in a qualitative manner the effects of a macromolecular factor purified from muscle conditioned medium on these neurons (J. P. Swerts, A. LeVan Thaï, A. Vigny, and M. J. Weber (1983) Dev. Biol. 100, 1-11; J. P. Swerts, Le Van Thai, and M. J. Weber (1984) 103, 230-234), raising the hypothesis of a common pathway in the regulation of neurotransmitter phenotype by these two agents.

Previous studies suggested the existence of two populations of cells in the mammalian sympathetic nervous system which differ in their ability to express phenylethanolamine N-methyltransferase (PNMT), the enzyme which specifically subserves the biosynthesis of epinephrine: (1) sympathoblasts and their progeny, the noradrenergic sympathetic neurons (PNMT negative); and (2) phaeochromoblasts , the precursors of the adrenergic cells of the adrenal gland and extra-adrenal chromaffin tissue (PNMT-positive). We sought to determine whether similar differences between sympathoblasts and phaeochromoblasts exist in other classes of vertebrate embryos. Using immunohistochemical and biochemical techniques to assay PNMT in sympathetic organs, we have found that chick embryo paravertebral ganglia contain PNMT activity both in vivo and in vitro. In vitro PNMT immunostaining was detected in principal neurons as well as in small process bearing neurons similar to mammalian SIF cells. In vivo, cells containing PNMT were seen not only in the adrenal gland, but also in other sympathetic structures such as the extra-adrenal chromaffin tissue and, unexpectedly, also in some cells of the kidney. Tyrosine hydroxylase, the first enzyme of the catecholamine (CA) biosynthetic pathway, was found in the same cells which could be stained with PNMT antibodies as well as in the sympathetic ganglia. Thus, we conclude that in contrast to the rat, chick sympathoblasts share with phaeochromoblasts the property of expressing all the CA enzymes, including PNMT.

Using a new coupled peroxidation method modified by adding nickel ammonium sulfate, we demonstrated the localization of monoamine oxidase (MAO) in the rat peripheral nervous system. MAO was localized in the endothelial cells of endoneurial vessels, in Schwann cell cytoplasm encircling myelinated axons, and in some unmyelinated axons. The morphometric ratio of these MAO-containing axons to the total unmyelinated axons was 10-13%. These MAO-containing unmyelinated axons were assumed to coincide with postganglionic sympathetic noradrenergic ones passing in the sciatic nerve. Histochemical MAO staining may be utilized to identify the postganglionic sympathetic nerves in normal and pathological conditions at the electron microscopic level in particular.

The activity of phenylethanolamine-N-methyl transferase (PNMT) has been measured in the chromaffin tissue of the head kidney and in the sympathetic neurons of the coeliac ganglion/splanchnic nerve in the cod. The content of adrenaline and noradrenaline in these tissues and in other adrenergically innervated tissues and blood plasma was also determined. Adrenaline dominates over noradrenaline in most sympathetically innervated tissues, in the chromaffin tissue and in blood plasma, but not in the muscularis mucosae of the swimbladder. High PNMT activity was found in the chromaffin tissue in the walls of the posterior cardinal veins, and also in sympathetic neurons. It is concluded that the adrenaline found in the sympathetic nerves may originate from intraneuronal adrenaline synthesis, but also from circulating adrenaline which is taken up and stored.

Secretin and vasoactive intestinal peptide (VIP) are known to stimulate tyrosine hydroxylase (TH) activity acutely in the rat superior cervical ganglion (SCG). Because TH-containing neurons in the SCG innervate the iris, submaxillary gland, and pineal gland, we examined the effects of secretin and VIP in these 3 autonomic end organs in vitro. Both peptides stimulated TH activity in each tissue. These stimulations resembled those in the SCG in that (1) secretin displayed a higher potency than VIP in all 3 end organs, (2) the peptide effects were unchanged when calcium was excluded from the incubation medium, and (3) they were mimicked by activators of the cyclic adenosine monophosphate (cAMP) pathway. These findings indicate that secretin and VIP can regulate transmitter metabolism in both the cell bodies and axon terminals of neurons originating in the SCG. Furthermore, the data raise the possibility that catecholamine synthesis in sympathetic nerve terminals is modulated by peptides released by other, nearby nerve endings.

In elucidating the morphologic basis of cervical spondylosis, the nerves accompanying the vertebral artery were studied under an operative microscope and by means of Karnovsky's histochemical method for acetylcholinesterase (AChE). The results show that the nerves arising from the cervical part of the sympathetic trunk and the cervical nerves anastomose with each other around the artery, and are distributed to the vertebral artery and the cervical spine. The nerves accompanying the vertebral artery show different degrees of AChE activity, and may be compressed or stimulated as a result of disease of Luschka's joint and the other degenerated structures, based on their anatomic relationship.

A differential assay for the determination of choline acetyltransferase activity in neuroblastoma is described, and the importance of using the differential assay is discussed. In addition, changes in cholinergic-adrenergic activity of cloned adrenergic neuroblastoma cells after serial transplantations are reported. The changes of the cholinergic activity could not be detected precisely without the use of the differential assay.

It has been postulated that the developing sympathetic innervation of rat eccrine sweat glands changes from adrenergic to cholinergic under the influence of its target. In agreement with previous evidence that the sympathetic innervation of adult rat sweat glands is cholinergic, we found that choline acetyltransferase (CAT)-immunoreactive nerve fibers are present in adult glands, and that gland-rich chunks of adult footpads contain CAT enzyme activity. We were therefore interested in determining when CAT activity is first expressed in the developing gland innervation. Low levels of acetylating activity were observed in rat footpads as early as postnatal day 4, when sympathetic fibers first contact the glands. A greater than fourfold increase in CAT specific activity occurred between postnatal days 11 and 21. Neonatal treatment of rats with the adrenergic neurotoxin 6-hydroxydopamine (6-OHDA) eliminated most of the CAT activity in 14 and 19 d footpads. In contrast, the acetylating activity observed prior to day 11 was unaffected by neonatal 6-OHDA treatment, and only slightly reduced by the selective CAT inhibitor, naphthylvinylpyridine. These results indicate that the sympathetic fibers that innervate rat sweat glands do not acquire detectable levels of CAT activity until a full week after they contact the glands.

E10 chick sympathetic ganglion cells display a cell contact-dependent rise in choline acetyltransferase (ChAT) specific activity over the first several days in culture. This effect can be mimicked by addition of crude membrane fractions prepared from E10 retina and adult chicken brain, but not by those from E10 brain. The effects of both cell-cell and membrane-cell contact are inhibited by the addition of anti-NCAM Fab fragments. The membranes capable of increasing ChAT and those which are ineffective all contain NCAM, however their relative levels of NCAM polysialic acid differ. Whereas membranes with high polysialic acid NCAM are ineffective, selective enzymatic removal of polysialic acid renders them capable of producing an increase in ChAT. The inhibition of NCAM-mediated adhesion produced by Fab fragments can be compensated for by addition of wheat germ agglutinin, but only with membranes whose NCAM has low levels of polysialic acid. Taken together, these data suggest that NCAM can regulate cell contact-mediated increases in ChAT activity. We propose that NCAM-mediated adhesion promotes contact between cell membranes to allow the transmission of an otherwise NCAM-independent signal. In addition, NCAM's polysialic acid moiety appears to influence the ability of cells to transmit this signal, even in the presence of an alternative adhesion mechanism.

Immature sympathetic ganglia prepared from 5 1/2-or 6-day-old chick embryos were cultured up to one month. The in vitro development was followed by phase microscopy, electron microscopy and using histochemistry for catecholamines, monoamine oxidase and cholinesterases. During the first week of culture extensive plexuses of nerve fibres were formed between and around the clusters of nerve cells. Mature-looking neurons were observed in the cultures by phase microscopy after three weeks, at which age the mean diameter of the perikarya was more than doubled. Varying catecholamine fluorescence was observed in the perikarya during the entire culture period. The nerve fibres showed usually only weak fluorescence, but, in the older cultures, bright varicosities were regularly found in the fibres. Monoamine oxidase activity was demonstrated already at three days of culture and the reaction was maintained positive. Weak or moderate acetyl-cholinesterase activity was demonstrated in the sympathicoblasts and young sympathetic neurons and their processes. The axolemma showed acetylcholinesterase activity also around the nerve terminals containing small dense cored vesicles. Reactions for the non-specific cholinesterases were negative. Electron microscopy of the 30-day-old cultures revealed that the clusters of nerve cells consisted of mature sympathetic neurons, which contained large (60-200 nm) and small (35-60 nm) granular catecholamine-storing vesicles. Glial cells were almost totally lacking. Large numbers of nerve terminals containing both large and small granular vesicles were observed in the clusters, often in synaptic contact with the sympathetic neurons. It is concluded that the primitive sympathicoblasts are, in favourable conditions, capable of differentiation in culture up to mature sympathetic neurons.

Immunocytochemical studies with a monoclonal antibody show that acetylcholinesterase (AcChoEase; EC 3.1.1.7) is distributed in clusters along the fibers of cultured sympathetic neurons but is essentially absent from cell bodies. Although tissue-cultured sympathetic neurons synthesize several oligomeric forms of AcChoEase, only the hydrophobic globular (G4) form of AcChoEase is present within these clusters. This G4 form is asymmetrically distributed within neurons and is transported preferentially into nerve fibers following its synthesis in the cell bodies. Thus G4 is found in clusters on neurons and is readily distinguishable from the hydrophilic forms on the surfaces of myotubes. The association of a specialized form of AcChoEase in densities on neurons in culture indicates that neurons and myotubes have distinct mechanisms for localizing AcChoEase molecules on their surfaces and suggests that these two types of electrically excitable cells have different requirements for organizing synaptic components on their plasma membranes.

The thiocholine method for the demonstration of AChE-containing fibres and the formaldehyde-induced fluorescence technique for the visualization of adrenergic fibres were employed to study the innervation of the albino rat iris. The following denervations were performed in order to verify the origins of different nerve types: (1) extirpation of the ciliary ganglion, (2) extirpation of the superior cervical ganglion, (3) stereotactic coagulation of the ophthalmic division of the trigeminal nerve, and (4) all possible combinations of the above-mentioned procedures. The denervations disclosed three main types of AChE-containing nerves in the iris: (1) nerve fibres degenerating after ciliary ganglionectomy, (2) thick nerve bundles in the dilator region disappearing after trigeminal neurotomy, and (3) fibres remaining intact after any type of denervation. Cervical sympathectomy had no effect on AChE-positive fibres. Under electron microscope AChE activity could be seen in the axolemma both in unmyelinated and in myelinated fibres. All fluorescent fibres vanished after ipsilateral cervical sympathectomy. Most of these fibres also disappeared after trigeminal neurotomy and the remaining fibres degenerated after subsequent ciliary ganglionectomy. On the basis of the present findings, the following conclusions can be drawn: (1) Most AChE-containing fibres of the rat iris originate in the ciliary ganglion. (2) The majority of the myelinated sensory fibres of the rat iris also contain AChE. (3) There is no AChE in the adrenergic fibres of the rat iris. (4) All adrenergic fibres of the rat iris originate in the ipsilateral superior cervical ganglion, and (5) these fibres enter the iris along with both the long and short ciliary nerves.

In this investigation we have combined the methods of ultrastructural demonstration of acetylcholinesterase activity with electron microscopic autoradiography for the demonstration of norepinephrine uptake. The results show electron-dense deposits indicative of acetylcholinesterase activity associated with perivascular axons overlaid by concentrations of silver grains representing exogenous tritiated norepinephrine. Forty-five percent of the intervaricose regions and 19% of the varicosities overlaid by autoradiographic grains showed "moderate" amounts of cholinesterase staining. A greater proportion of autoradiographic grains was observed on the varicosities than in the intervaricose regions; however, the amount of acetylcholinesterase activity was greater in the intervaricose regions than in the varicosities. This investigation provides evidence for the presence of periaxonal acetylcholinesterase staining in adrenergic axons in the rat kidney.

There is first evidence that the adult human pineal gland may be equipped with noradrenergic fibers as is the case in a variety of mammalian species. It therefore appears worthwhile to investigate the capacity of pineal noradrenergic nerves to take up 123I-MIBG, a prerequisite for scintigraphic imaging.

By means of the AChE in toto staining method (Baljet and Drukker 1975) the peripheral nervous system in several human fetuses has been investigated. Especially concerning the peripheral autonomic nervous system some striking results were found. In the human fetus there exists a large variability in number and diameter of the communicating rami. Many ganglia in the sympathetic trunks are fused. The major splanchnic nerve arises at various levels from the thoracic sympathetic trunks as well as many smaller thoracic sympathetic nerves. Furthermore various visceral nerves and nerve related elements are presented in this study.

The molecular forms of acetylcholinesterase (AcChE) have been studied in primary cultures of newborn rat sympathetic neurons grown either in the absence (CM- cultures) or in the presence (CM+ cultures) of muscle conditioned medium. The cultures were treated with a mitotic poison to eliminate non-neuronal cells. CAT activity increased with time in culture 4- to 20-fold faster in CM+ than in CM- cultures. In agreement with previous experiments (J.P. Swerts, A. Le Van Thaï, A. Vigny, and M.J. Weber, 1983, Develop. Biol. 100, 1-11), AcChE activity developed at a 3-fold lower rate in CM+ than in CM- cultures. This deficit in AcChE activity in CM+ cultures resulted from a deficit in the number of enzyme molecules immunoprecipitable with an antiserum raised against rat brain AcChE. In both types of cultures, AcChE forms were separated by sucrose gradient sedimentation into three main peaks corresponding to the 16 S and 10 S forms and a mixture of the 6.5 and 4 S forms. In 3-day-old CM+ and CM- cultures, the 16 S form represented 2% of the total activity. After 12-26 days, the percentage of 16 S form raised to 15-30% in CM- cultures, but remained lower than 5% in CM+ cultures. This difference was also observed when AcChE molecular forms were analyzed in the presence of protease inhibitors. A similar result was obtained by comparing cultures grown with and without a macromolecular factor partially purified from conditioned medium. These results suggest that an inverse relationship exists between the presence of 16 S AcChE and the presence of cholinergic synapses in these cultures.

Glucocorticoids stimulate tyrosine hydroxylase activity and catecholamine synthesis, while markedly inhibiting acetylcholine synthesis and storage in the a clone of sympathetic nerve-like cells. Nerve growth factor enhances the effect of glucocorticoids on tyrosine hydroxylase. The steroid effect is specific for glucocorticoids, since 11-desoxycortisol, testosterone, and estradiol-17 beta do not reproduce the effects of hydrocortisone and dexamethasone. Concomitant with the shift in neurotransmitter synthesis, there is an increase in the mean diameter of intracellular dense core vesicles. In contrast to glucocorticoids, insulin increases the specific activity of choline acetyltransferase through the interaction with typical insulin receptors. Insulin does not, however, alter the morphology of the cells, nor does it block the morphological response of the cells to nerve growth factor.

The soluble proteins isolated from sympathetic nerves were separated on Fractogel TSK-HW columns. With a mobile phase of 0.1 M phosphate + 0.1 M K2SO4, pH 6.8, the main fractions I-VI were obtained. These fractions were analysed by high-performance (HPLC) on TSK-SW columns. Fractogel fractions I-III showed peaks of molecular weights, Mr670,000, as estimated by HPLC. With sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) these fractions show no bands stainable with Coomassie Blue. The protein of fraction IV was L-DOPA decarboxylase (AADC E.C. 4.1.1.28) with Mr 150,000 existing of subunits with Mr 55,000, 45,000, 27,000 and purified according to Christenson et al. (Arch. Biochem. Biophys., 141 (1970) 356). The dopamine-beta-hydroxylase (E.C. 1.14.2.1) subunits with Mr 75,000 proteins were detected in Fractogel fraction V. Fraction VI was Mr 27,000 protein. Proteins with molecular weights Mr less than 5,000 were also detected. With Phenothiazine-Affigel the proteins of fraction V (mr 75,000) showed no affinity to the phenothiazine column equilibrated with application buffer containing Ca2+ X 50-70% fraction IV (Mr 150,000), eluted with Tris-EGTA buffer, and 100% fraction VI (Mr 27,000) showed affinity to the Phenothiazine-Affigel column.

The localization of L-glutamate decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid, was studied in newborn rat retroperitoneal sympathetic tissue, i.e. the main retroperitoneal paraganglion, adrenal medullae and abdominal sympathetic ganglia using the indirect immunofluorescence method. The coexistence of GAD with the catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) was analyzed in consecutive sections or by staining one section consecutively with different antisera. GAD immunoreactivity was observed only in some cell types of each organ studied. In the main retroperitoneal paraganglion, the small, intensely TH-immunoreactive, paraganglion-type cells were GAD-immunoreactive, while the larger moderately TH-immunoreactive, neuron-like cells were non-reactive for GAD. In the adrenal medulla, GAD immunoreactivity was localized only in the adrenaline-synthesizing, PNMT-immunoreactive chromaffin cells. The noradrenaline-synthesizing, i.e. the TH-immunoreactive cells with no PNMT immunoreactivity, were non-reactive for GAD. In the abdominal sympathetic ganglia, some small intensely TH-immunoreactive cells were GAD-immunoreactive, while the principal neurons were non-reactive for GAD. These results provide immunohistochemical evidence that GAD is present and is colocalized with catecholamine-synthesizing enzymes in various sympathetic tissues of the newborn rat. The present results indicate that GAD is localized in adrenaline-synthesizing cells of all the sympathetic tissues studied. A fraction of noradrenaline-synthesizing cells of retroperitoneal sympathetic tissues, excluding the adrenal medulla, also contains GAD.

Damage to the rat septohippocampal pathway results in the growth of sympathetic axons from nearby blood vessels into the denervated hippocampal formation. Sympathohippocampal sprouting exhibits lesion specificity--that is, only injury to the septohippocampal projection elicits the sprouting response. Whether other perivascular fibers sprout in response to septohippocampal injury (response specificity) has been addressed in the present study. Using cathecholamine histofluorescence and acetylcholinesterase histochemical techniques, we determined the distribution and incidence of perivascular sympathetic and nonsympathetic fibers associated with parahippocampal blood vessels in normal rats and in rats sustaining medial septal lesions. We found that sympathetic fibers are more numerous than acetylcholinesterase-positive fibers at all septotemporal levels of the hippocampal formation and that both types are very rare at dorsal hippocampal levels in normal rats. Following medial septal lesions, however, there is a tremendous increase in the number of perivascular sympathetic fibers at dorsal hippocampal levels but no change in the number of acetylcholinesterase-positive fibers. Electron microscopic observations indicate that the increase in perivascular fibers is due to increases in the number of sympathetic axonal fascicles as well as the number of axons per fascicle. Furthermore, both light and electron microscopic data suggest that parahippocampal veins are normally not accompanied by perivascular fibers but are associated with sympathetic fibers following medial septal lesions. These results indicate that sympathetic sprouting in response to septohippocampal denervation exhibits specificity not only in terms of the lesion which elicits such sprouting but also in terms of the types of fibers that respond to the lesion.

A procedure is described for measuring the activity of tyrosine hydroxylase in the intact adrenergic neurons of the mouse vas deferens. In this procedure, the L-dopa-1-14C which is formed from L-tyrosine-1-14C by the action of tyrosine hydroxylase is selectively and quantitatively decarboxylated by endogenous aromatic-L-amino acid decarboxylase. Although considerable tyrosine hydroxylase activity can be demonstrated with the intact mouse vas deferens in the absence of exogenous tetrahydropterin cofactor, the addition of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine results in increased activity. Exogenous norepinephrine inhibits the activity of tyrosine hydroxylase in the intact mouse vas deferens and this inhibitory effect is competitively antagonized by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. When the vesicular catecholamine uptake mechanism is blocked by treatment of mice with 5 mg/kg of reserpine 24 hours prior to sacrifice, the activity of tyrosine hydroxylase in intact vas deferens is reduced and the inhibitory effect of exogenous norepinephrine is enhanced. Inhibition of monoamine oxidase with 0.15 mM pargyline does not affect the activity of tyrosine hydroxylase in the intact mouse vas deferens when the vesicular catecholamine uptake mechanism is intact but has a pronounced inhibitory effect following reserpine treatment. These observations lend further support to the conclusion that the activity of tyrosine hydroxylase in the intact adrenergic neuron is inversely related to the catecholamine concentration within an extravesicular pool. They also suggest that the catecholamines tend to accumulate within this extravesicular pool and thus become accessible to the action of monoamine oxidase when the vesicular uptake mechanism is inactivated or when the vesicular stores are filled to capacity.

The purpose of the studies reviewed here was to investigate the role of inheritance in the regulation of human serum dopamine-beta-hydroxylase (DBH) activity. DBH is a catecholamine biosynthetic enzyme that is localized to catecholamine-containing vesicles, is released with catecholamines from sympathetic nerves and the adrenal medulla, and is found in serum. It has been suggested that serum DBH activity might be a useful and convenient measure of sympathetic nervous system function. DBH activity was measured in blood from large, randomly selected populations of children, adolescents, and adults and in blood of relatives of children with very low serum DBH activity (less than 50 units). The results of sibship and pedigree analyses of data from families of probands with very low enzyme activity were compatible with autosomal-recessive inheritance of very low serum DBH activity. In addition, the results of studies of immunoprecipitable serum DBH were compatible with a genetically mediated decrease in the quantity of DBH protein in the blood of subjects with this allele. No significant correlation of serum DBH activity with either systolic or diastolic blood pressure was found in a large, randomly selected population of children.

Disturbances of central catecholaminergic-cholinergic balances have been discussed as causing affective disorders and schizophrenia. Such imbalances might be due to abnormalities of the metabolizing enzymes, especially their activities relative to each other. With this in mind, the activities of platelet monoamine oxidase and plasma butyrylcholinesterase (pseudocholinesterase) were determined spectrophotometrically in 33 psychiatric patients and eight controls. No significant differences could be detected for the enzyme activities as such and their relationship as expressed by their ratios. Thus, these peripheral enzymes seem to be unlikely indicators of supposed central imbalances.

By using a new microdissection procedure allowing the noradrenaline (NA) and adrenaline (A) cell groups of the A2-C2 region to be sampled preferentially, it was possible to study the biochemical response of these two neuronal populations after 6-hydroxydopamine (6-OHDA) administration. Five days after an intraventricular 6-OHDA injection, tyrosine hydroxylase (TH) activity increased (+104%, P less than 0.01) in the adrenergic C2 region, in the locus coeruleus (LC) and in the A1-C1 region, while the NA A2 region exhibited no significant increase. Twenty-one days after 6-OHDA administration, dopamine-beta-hydroxylase (DBH) activity had decreased in both the noradrenergic regions (LC, A1-C1 and A2 regions) and in the C2 adrenergic region. Conversely, phenylethanolamine-N-methyltransferase (PNMT) activity was not modified either in the cell bodies or in the terminals located in the tractus intermediolateralis of the spinal cord and in the hypothalamic nuclei. These data suggest: (i) that adrenaline-containing neurons could be sensitive to the neurotoxic action of 6-OHDA since they exhibit changes in TH and DBH activities; and (ii) that the determination of PNMT activity may not be sensitive enough to estimate the functional integrity of the A cell bodies or terminals.

Compared with the myocardium, glycolytic enzymes are reduced by 50% and mitochondrial enzymes and space by 70% in the conduction system of the calf heart. In addition, on the basis of adenosine triphosphate activities energy demands are reduced by more than 50%; this is in parallel with the reduction in myofibrillar space. The increased tolerance of the conduction system against ischemia can be explained by a reduction of energy demands and a higher proportion of (anaerobic) glycolytic as opposed to aerobic mitochondrial energy production. Among the structures of the conduction system, the sinoatrial and atrioventricular nodes are markedly susceptible to hypoxia in contrast to atrial conduction and ventricular conduction by way of the His-Purkinje system. In the isolated perfused rat heart, an increased net release of noradrenaline during the first 10 minutes of ischemia is only noted after sympathetic stimulation. During this phase, catecholamine overflow is limited by the activity of the neuronal reuptake. At a later second phase, from 15 to 40 minutes after the onset of ischemia, the mechanism of noradrenaline net release is carrier-mediated efflux inhibited by neuronal uptake blocking agents. During the third phase of ischemia, after about 40 minutes, spontaneous noradrenaline release is greatly augmented, probably as a result of leakage caused by membrane damage.

Dopamine-beta-hydroxylase (DBH) activity in serum, DBH and tyrosine hydroxylase (TH) activities in mesenteric vessels, and DBH and TH activities in locus coeruleus and hypothalamus of brain did not differ significantly between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKR) at 16 weeks of age when hypertension of SHR was fixed. In contrast, DBH and TH activities in vas deferens and adrenal glands were significantly higher in SHR than in WKR. These changes in SHR at 16 weeks of age after establishment of hypertension are directly opposite those reported previously in SHR at 3 weeks of age before the onset of hypertension.

Highly purified sympathetic nerve vesicles isolated from bovine splenic nerves were treated by hypo-osmotic shocks, freeze-thawing or incubation in the absence or presence of ATP and MgCl. The vesicle preparations were then studied morphologically by electron microscopy and their content of noradrenaline (NA), and soluble proteins analyzed biochemically with special regard to dopamine beta-hydroxylase (DBH). Hypo-osmotic shocks released about 25 per cent of the NA and protein content and about 8 per cent of the DBH activity. This treatment induced swelling of the vesicles but their membranes remained unruptured and they still contained dense cores. Freeze-thawing released about 35 per cent of the NA, 25 per cent of the proteins and 11 per cent of the DBH. After the latter treatment some matrix material still remained in most vesicles but many were less stainable than the intact vesicles in cold control preparations. During incubation at 30 degrees C in an isotonic sucrose-phosphate medium for 30 min the vesicles released most of their NA and soluble DBH activity as well as much of their matrix density. After incubation at 37 degrees C for 30 min most vesicles appeared translucent. After incubation at 30 degrees C for 30 min in the presence of ATP and MgCl the vesicles lost most of their original NA content but retained their DBH activity and most of their matrix density. The results indicate that there is not always a correlation between NA content and retention of matrix density which suggests that DBH might be a component of a macro-molecular complex responsible for the staining reaction taking place in the maxtrix of NA depleted vesicles. This hypothesis is further supported by the finding of striking similarities between DBH isolated from chromaffin granules and the granular and fibrillar material surrounding the nerve vesicles after depletion.

The peroxidase-antiperoxidase (PAP) technique is used to determine the cellular localization of tyrosine hydroxylase in the human fetal nervous system. Antiserum to trypsin-treated tyrosine hydroxylase from the bovine adrenal medulla can be detected immunocytochemically in peripheral sympathetic neurons in the 8-mm (5-week) fetus, but can not be detected in the central nervous system until later stages of development. The cytological features and distribution of the neuronal perikarya and processes labeled for the enzyme are similar to those of the catecholaminergic neurons previously identified by histofluorescence. These findings indicate that specific neurons in the human fetus have at least one of the enzymes necessary for the biosynthesis of catecholamines and that cross-species reactivity exists between antiserum produced to the bovine tyrosine hydroxylase and human tissues.

In this study the development of vagus nerves and the development of sympathetic nerves related to the development of the upper gastro-intestinal tract was studied in the rat from 12.5 days p.c. until birth by means of enzyme histochemical methods applied to sections and toto preparations. A striking time relation between the ingrowth of the vagus nerves as well as the sympathetic nerves and the appearance of acetylcholinesterase (AChE)-positive cells in the wall of the upper gastro-intestinal tract is established. The maturation of the upper enteric ganglion cells is dependent on the presence of a vagal influence. The vagal ingrowth of the stomach starts at about day 12.5 p.c. In AChE toto preparations it is established that the basic distribution pattern as described in the adult rat is complete at day 15 p.c. However, in contrast with the adult state, gastric branches can be traced up to the pylorus and the greater curvature until day 18 p.c. During the 18th day p.c. there is a tremendous increase in the surface area of the stomach. This increase in surface area of the gastric wall and relative decrease in vagal outgrowth leads to the adult configuration of vagal ramifications in relation to the wall of the stomach which can already be observed on the 19th day p.c.

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.

1 No statistically significant difference in the activity of choline acetyltransferase (ChAT) was detected between sympathetically denervated and control rabbit ear artery (REA) tissue. This was interpreted as evidence against the hypothesis that endogenous acetylcholine plays an obligatory role in sympathetic postganglionic neurotransmission. 2 The values obtained for ChAT activity in the extraneuronal REA tissue were very low, but were greater than the boiled blank values. 3 Treatment with a specific inhibitor of ChAT did not reduce the REA values, while it did for rabbit iris. Addition of acetylcholinesterase to the REA assay reduced the activity of the collectable product to a markedly lesser degree than was observed with other tissues. 4 The specificity of the enzyme assay at the very low yield levels observed in the extraneuronal REA tissue was therefore questioned.