Biomedical subjects
Publications and source records attributed to M Schultzberg.
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By using the Coons indirect immunofluorescence technique, enkephalin-like immunoreactivity with a granular localization was observed in human adrenal medullary gland cells and pheochromocytomas. In two of the tumors and in a few adrenal gland cells, a somatostatin-like peptide could also be identified. Catecholamine cell types were visualized on adjacent sections with antisera to the synthesizing enzymes dopamine-beta-hydroxylase [DBH; dopamine beta-monooxygenase; 3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] and phenylethanolamine-N-methyltransferase (PNMT; noradrenalin N-methyltransferase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28). In the normal adrenal medulla more DBH- than PNMT-immunoreactive gland cells were observed. In the adrenal pheochromocytoma both DBH- and PNMT-positive cells were seen, whereas the two extra-adrenal tumors contained only DBH. These findings correlated well with plasma catecholamine measurements. Finally, enkephalin immunoreactive fibers and somatostatin immunoreactive cells were observed in a sympathetic ganglion extirpated together with one of the tumors.
Small intestine from 18-day fetal mice grown for 3 weeks in organotypic tissue culture was found to contain numerous VIP, enkephalin, substance P and some somatostatin immunoreactive nerve fibers. Since these cultures should be devoid of all afferent or other extrinsic neuronal inputs, it is concluded that there are VIP, enkephalin, substance P and somatostatin containing neurons intrinsic to the intestinal wall. However, all 4 peptides may also be present in neurons originating outside the gastrointestinal tract as well as in the intrinsic neurons.
The localization of substance P (SP) or a SP-like peptide in cultured ganglia from chick embryos was studied by the indirect immunofluorescence technique. Ganglia from 8-16 days old chick embryos and from newly hatched chickens were cultured in a control medium or in the presence of nerve growth factor (NGF). Addition of colchicine and exposure to different explanted peripheral tissues were also tried. Ganglia from the younger embryos (8-12 days) cultured for 24 h with added NGF showed a weak SP-like immunoreactivity (SPLI) in some cell bodies and strong specific immunofluorescence in nerve fibres growing out from the ganglia. In spinal ganglia of the older embryos (14 and 16 days) and newly hatched chickens cultured with and without NGF the concentration of SPLI in the cell bodies was considerably higher. Addition of colchicine to spinal ganglia cultured 12 h in NGF-medium, resulted in retraction of nerve fibres and strongly fluorescent, expanded nerve fibres were observed in peripheral parts of the ganglia. Explants of skin placed near the spinal ganglia stimulated the outgrowth of fibres, some of them containing SPLI. A few fluorescent fibres were also seen within the skin explants. Also heart tissue explants stimulated outgrowth of nerve fibres, but innervation of these explants with SPLI-containing nerves could not be observed. Nerve fibre-extension from the spinal ganglia was not stimulated by spinal cord explants. The present results support the existence of SP-containing primary sensory neurons in chickens.
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Using the indirect immunofluorescence technique of Coons and collaborators, neurons containing substance P-, enkephalin-, vasoactive intestinal polypeptide (VIP)--and somatostatin-like immuno-reactivity have been identified in the peripheral nervous system. They have a widespread distribution, particularly in the gastrointestinal and urinary tracts. Whereas part of these peptide containing fibres may belong to sensory neurons, the majority seem to have their origin in peripheral autonomic ganglia, indicating a complex built up of the autonomic nervous system. There is evidence that some noradrenergic neurons contain somatostatin, which may suggest that one neuron can synthesize and store two transmitters. The significance of such neurons, as well as of peripheral peptide neurons in general, remains to be elucidated.
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Several sympathetic ganglia of the guinea pig, cat and rat were studied with indirect immunofluorescence technique using antibodies to substance P (SP) and dopamine-beta-hydroxylase (DBH), the latter of which represents a marker for noradrenaline containing neurons. In all ganglia studied SP-like immunoreactivity could be observed in certain nerve fibers (SP-positive fibers). Their number, localization fluorescence intensity and 'fluorescence morphology' varied considerably between the different ganglia as well as between species. In the inferior mesenteric ganglion and the coeliac-superior mesenteric ganglion complex of all species, and in particular of the guinea pig, a dense plexus of varicose SP-positive fibers was observed around the mostly DBH-positive, principal ganglion cells. In contrast no such fibers were seen in relation to the DBH-positive SIF cells of the ganglia. No SP-positive ganglion cells were observed in any of the ganglia studied. Previous biochemical and immunohistochemical studies have indicated that peripheral SP-positive fibers may represent the branches of primary sensory neurons. The rich supply of varicose, SP-positive nerve terminals, often surrounding the noradrenergic ganglion cells in a basket-like manner, may indicate that these fibers may play a functional role within the ganglia. Instead of merely passing through the ganglion they may be involved in special types of reflex arches.
By means of the indirect immunofluorescence technique of Coons and collaborators, somatostatin-like immunoreactivity has been demonstrated in principal ganglion cells of some sympathetic ganglia. The noradrenergic nature of these cells was established by "staining" of the same or consecutive sections with antiserum to dopamine beta-hydroxylase [dopamine beta-monooxygenase; 3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1], the enzyme converting dopamine to noradrenaline (norepinephrine). In guinea pigs the somatostatin immunoreactive material was found in almost two-thirds of all principal ganglion cells of the coeliac-superior mesenteric ganglion complex (anterior inferior part) and of the inferior mesenteric ganglion, but only in a few cells of the superior cervical ganglion. It appeared to be localized close to the Golgi complex. The present findings may represent a concomitant storage of a biogenic amine and a small peptide in a neuron. Because both noradrenaline and somatostatin may fulfill a role as a neurotransmitter or modulator, the sympathetic neurons described in this study may represent an example of mammalian nerve cells not conforming to Dale's hypothesis, i.e., the one neuronone transmitter concept.
A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3',5'-cyclic nucleotide phosphodiesterase, and (Na+ + K+)-ATPase. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A2 attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A2 was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3',5'-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500-4000A), which do not form vesicles.
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The present study demonstrates the occurrence of substance P (SP)- and calcitonin gene related peptide (CGRP)-immunoreactive nerve fibres in bone, bone marrow, periosteum, synovial membrane and soft tissues adjacent to the bone. The distribution pattern of the two types of nerves was similar, although the CGRP-positive fibres generally were more numerous. Both types of nerves were particularly abundant near the epiphyseal plate, in the bone marrow of patella and epiphyses, and in the periosteum. Many SP- and CGRP-immunoreactive fibres were also observed around blood vessels.