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In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

A sodium-independent neutral and basic amino acid transporter (NBAT) from rat kidney was recently cloned and its amino acid sequence deduced. We used light and electron microscopic immunoperoxidase labeling to determine the cellular localization of NBAT in rat kidney and small intestine. The localization was carried out using site-directed antisera raised against synthetic peptides within NBAT. The most prominent localization of NBAT was in microvilli of epithelial cells lining renal proximal tubules. Microvilli of small intestinal epithelia were less frequently immunoreactive. Unexpectedly, the most intense labeling in the small intestine was seen within enteroendocrine cells and submucosal neurons. The neuronal labeling was highly localized within dense core vesicles in axon terminals apposed to the basal lamina near fenestrated blood vessels. These results support the proposal that NBAT plays a role in reabsorption of amino acids in renal tubules. In addition, they suggest that NBAT (or NBAT-like proteins) may have multiple functions in the small intestine, including luminal uptake of amino acids and vesicular uptake of related substrates into enteroendocrine cells and enteric neurons.

We sought to determine whether the precursors of catecholamine-containing neurons in the developing peripheral and central nervous systems of chickens and rats express the biosynthetic enzymes tyrosine hydroxylase [THase; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] or dopamine beta-hydroxylase [DBHase; 3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1], prior to the time they withdraw from the cell cycle. Chicken embryos (stages 26-27) were injected with [3H-thymidine and 4 hr later were prepared for the simultaneous demonstration of radioautographically labeled nuclei in immunoreactive THase cells. The brains and sympathetic chains of rat fetuses (days E12-E14), exposed for 2 hr to [3H]thymidine, were treated similarly except that peripheral tissues were stained with a specific antibody to DBHase as well as anti-THase. In the peripheral nervous system of both chicken and rat, nuclei of THase-containing cells were radioautographically labeled. DBHase-containing cells in the peripheral nervous system of rats were also labeled and thus are noradrenergic. THase was localized in cells of the brain of the same rat fetuses beginning on day E12 (no THase was detected on day E11 or E11.5) in the mantle layer of the ventral mesencephalic and rostrolateral rhombocephalic cellular groups; however. THase-containing cells in the central nervous system did not incorporate [3H]thymidine. We conclude that, during development, the adrenergic neuronal precursors of the peripheral nervous system but not of the central, have the capacity to synthesize catecholamines before they withdraw from the cell cycle. Differences in the maturation of peripheral and central neurons may be related to differences in their embryological origin.

A specific antibody to tryptophan hydroxylase [L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4] has been used to localize the enzyme immunohistochemically in neurons of the mammalian gut. The enzyme was found in perikarya of intestinal neurons of mice, rats, and guinea pigs. Neurons containing the enzyme survived for up to 3 weeks in organotypic tissue culture and were intrinsic to the gut. These neurons are probably serotonergic and are the first such neurons to be found in the peripheral nervous system.

The distribution of dopamine-containing processes in the striatum of fetal and neonatal cats was studied by immunohistochemical and glyoxylic acid histofluorescence methods and compared to the distribution of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) observed by thiocholine histochemistry in the same or serially adjoining sections. Both methods for demonstrating the dopamine innervation revealed the characteristic patchwork of dopamine "islands" in the caudoputamen, in which catecholamine histofluorescence or tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]-like immunoreactivity was concentrated into 0.2- to 0.6-mm-wide patches. Both methods also demonstrated a high degree of patterning of the dopamine innervation in the ventral striatum, including the nucleus accumbens septi. A detailed and striking match was found between these configurations and the compartmental distribution of acetylcholinesterase observed in the caudoputamen and ventral striatum of the same brains. The correspondence between the dopamine and acetylcholinesterase figures was most obvious in the fetal brains, in which the background acetylcholinesterase staining was lightest, but matches between the dopamine islands and acetylcholinesterase patches could still be seen in the kittens. There was no clear alignment of striatal cell bodies stained for acetylcholinesterase with either the dopamine or the acetylcholinesterase-positive patches. Nor was there an obvious correspondence between dopamine and acetylcholinesterase in the striatal background matrix. We conclude that, at least during ontogenesis, it is the clustered arrangements of dopamine and acetylcholinesterase that are, in particular, tightly linked, and we suggest that information about the maturation of these clusters may be crucial in assessing the functions of striatal dopamine and acetylcholinesterase in the adult.

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from the region of the raphe nucleus of rat midbrain by sequential column chromatography and disc-gel electrophoresis. In electrophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to electrophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chlorohenylalanine. Antibodies produced to each of the three bands crossreacted by immuno double diffusion and electrophoresis with each other and homogenates of raphe nuclei; they completely inhibited enzyme activity only of tryptophan hydroxylase. Tryptophan hydroxylase was localized by light and electron immunohistochemistry to serotonin neutrons of the raphe. Ultrastructurally, in cell bodies, the enzyme was distributed in cytoplasm and in association with endoplasmic reticulum and Golgi apparatus. In dendrites and axons, it was associated with microtubules. Tryptophan hydroxylase in brain is only neuronal and cytoplasmic, exists in multiple forms, and is associated with microtubules, suggesting it may be transported from sites of synthesis in cell body into axons.

Tyrosine hydroxylase (EC 1.14.16.2), the enzyme catalyzing the rate-limiting step in catecholamine biosynthesis, was localized by electron microscopy within noradrenergic neurons of the nucleus locus coeruleus of the rat brain with a specific antibody to tyrosine hydroxylase labeled by the peroxidase-antiperoxidase immunohistochemical method. Labeled cell bodies and their processes were easily distinguished from unstained neuronal elements in the neuropil. The hydroxylase was only seen in the neuronal cytoplasm. Its distribution in processes was different from that in cell soma. In longitudinal sections of axons and dendrites, the peroxides reaction product appeared as fiber-like strands aligned parallel with the plasma membrane. In cross section, the labeled structures had a diameter of 220 A and exhibited an orderly distribution within the processes. The size and distribution of the peroxidase-stained structures suggest that they are neurotubules. In the perikarya, the cytoplasm was diffusely stained; the reaction was most intense on membranes of endoplasmic reticulum and Golgi apparatus, whereas lysosomes and mitochondria did not stain. The ultrastructural localization of tyrosine hydroxylase is consistent with biochemical data suggesting that the enzyme exists in different states in cell body and processes. The ultrastructural identification of enzymes subserving synthesis of neurotransmitters in central neurons and their processes may provide a useful tool in mapping the distribution of chemically specific synapses on identifiable neurons in brain.

Antibodies to the beta 2-adrenergic receptor of frog erythrocytes have been raised in rabbits by immunization with purified receptor preparations. Binding of the antibodies to the receptors was demonstrated by immunoprecipitation and by the altered mobility of the antibody-bound receptors on steric-exclusion HPLC columns. As assessed by a radioimmunoassay developed with the antibody, beta 2-adrenergic receptors from several sources showed various degrees of immunological crossreactivity whereas several beta 1-adrenergic receptors did not crossreact. The antibody appeared to not bind at the ligand binding site of the receptor and did not perturb antagonist radioligand binding to the receptor. Nonetheless, the antibodies selectively attenuated catecholamine-stimulated adenylate cyclase. This suggests that the antibodies recognize and bind to domains of the receptor other than the binding site and that may be involved in coupling to other components of the adenylate cyclase system. Immunocytochemical techniques were used with the antibodies to delineate a postsynaptic localization of beta-adrenergic receptors in rat and frog brain. Thus, these anti-beta-adrenergic receptor antibodies provide a useful reagent for probing beta-adrenergic receptor structure, function, and localization.