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ABSTRACT: Co-storage of enkephalins and catecholamines in coronary artery, mesenteric artery and vein, middle cerebral artery, vas deferens and adrenal medulla was studied in domestic pig (Sus scrofa). Responses to acute CNS ischemia were correlated with time to peak plasma levels of central venous and adrenal vein outflow samples in controls, during reserpine treatment and after drug withdrawal. Endogenous enkephalins are co-stored in chromaffin granules of adrenal epinephrine-type cells and large dense cored vesicles of noradrenergic terminals. After a lag period, reserpine at near 'therapeutic' doses caused an apparent induction of opioid peptide precursor synthesis accompanied by processing to enkephalins in adrenal medulla up to 8-fold by 30 days and in mesenteric vein up to 4.5-fold by 14 days. Upon 14 days recovery from reserpine, elevated adrenal enkephalins were maintained and depleted catecholamines were largely replenished. Acute CNS ischemia produced rises in MAP (approx. 80 mmHg), marked net depletions of noradrenergic enkephalin stores, and net increases in adrenal vein outflow and central venous levels of enkephalins and catecholamines. Noradrenergic terminals contributed significantly to circulating enkephalins as well as norepinephrine. Reserpine for 7 days nearly abolished all tested responses to acute CNS ischemia, but immediate net 200-400% elevations of endogenous enkephalin stores occurred in coronary artery and mesenteric artery and vein (apparent processing of reserpine-induced neuronal precursor stores). Thus, induction of new synthesis of precursor opioid peptides by reserpine, with or without parallel processing to enkephalins, occurs in noradrenergic terminals in many tissues. All effects of reserpine on endogenous enkephalins implicate a central mechanism to inhibit sympathoadrenal outflow to the periphery. At 14 days recovery from reserpine, when near normal cardiovascular responses to acute CNS ischemia were regained, there was increased net release of the elevated adrenal enkephalins, exaggerated peak plasma enkephalin concentrations, but only minimal depletions of enkephalins from noradrenergic terminals.
Journal of the Autonomic Nervous System 05/1990; 30(1):37-62.
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Blood vessels 02/1989; 26(3):177-84.
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ABSTRACT: The effect of substance P on leukocytes and platelets in rabbit skeletal muscle microvasculature was studied by intravital microscopy and electron microscopy. Local application of substance P caused vasodilatation and formation of aggregates of platelets and leukocytes in postcapillary venules with subsequent migration of leukocytes through the vessel wall. Many neutrophils were partially degranulated. Aggregate formation induced by substance P could not be prevented by autacoid antagonists. However, superfusion with calcitonin gene-related peptide prior to challenge with substance P greatly inhibited aggregate formation and leukocyte extravasation.
Blood vessels 02/1989; 26(2):84-94.
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ABSTRACT: A differential distribution of vasoactive neuropeptides and serotonin in chromaffin cells and nerve fibers within the adrenal glands of the pig (Sus scrofa) was found using immunohistochemical methods. Met- and leu-enkephalins, present at high levels in the medulla (measured by radioimmunoassay), occurred in adrenaline storing cells, some of which contained calcitonin gene-related peptide. Islets of chromaffin cells beneath the capsule also contained enkephalins and calcitonin gene-related peptide. Nerve fibers with enkephalin-like immunoreactivity were sparse, but many varicose fibers in the inner cortex and medulla showed calcitonin gene-related peptide immunofluorescence in a pattern similar to vasoactive intestinal polypeptide. Neuropeptide Y was mainly associated with perivascular fibers and neither neuropeptide Y nor vasoactive intestinal polypeptide immunoreactive chromaffin cells were detected. In contrast to the neuropeptides, most serotonin-like immunoreactivity coincided with noradrenaline histofluorescence. It is concluded that the distribution of nerve fibers with calcitonin gene-related peptide and vasoactive intestinal polypeptide would allow interactions between chromaffin and inner cortical cells. Stimuli activating noradrenaline chromaffin cells could release serotonin while stimulation of adrenaline storage cells would release enkephalin and, to a lesser extent, calcitonin gene-related peptide. Met-enkephalin, which occurs 3 4:1 over leu-enkephalin, is the most likely of the co-released peptides to reach distant receptors via the venous outflow.
Neuroscience 02/1989; 28(3):765-75. · 3.38 Impact Factor
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Acta Physiologica Scandinavica 01/1989; 134(4):579-80. · 2.55 Impact Factor
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ABSTRACT: The distribution of substance P (SP)- and neuropeptide Y (NPY)-immunoreactive (IR) nerve fibers in the rabbit tenuissimus muscle was investigated by means of immunohistochemistry. Electron microscopy was used to study the ultrastructural appearance of nerve fibers and terminals. SP-IR nerve fibers were sparse in the main feeding vessels to the muscle and in the central artery and vein, but moderately dense in the motor nerve and in nerve bundles running in the vicinity of the vessels. Occasionally, fibers were seen in apposition to arterioles and venules in the muscle. NPY-IR nerves formed a dense network of a typically adrenergic appearance encircling the feeding artery, central artery, and arterioles of all sizes. NPY-IR nerves were not seen around venules or veins. In the motor nerve, NPY immunoreactivity could be seen after ligation. Electron microscopy showed nerve terminals containing both small vesicles and large dense core vesicles outside the media of arterioles and, more seldom, of venules. Also, unmyelinated fibers followed myelinated nerve bundles along arterioles. The fact that there are a great many SP- and NPY-immunoreactive fibers in the tenuissimus muscle, with a distribution that harmonizes with their pharmacological actions, supports the view that local release of these neuropeptides contributes significantly to microvascular regulation in skeletal muscle.
Microvascular Research 10/1988; 36(2):117-29. · 2.83 Impact Factor
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ABSTRACT: The functional states of cerebral perivascular noradrenergic terminals were investigated following experimental 'closed-space' subarachnoid haemorrhage (SAH) in cat. The left middle cerebral artery (L-MCA) was compared to the ruptured right (R-MCA) one. Permeability kinetics of 3H-NA (noradrenaline) were measured simultaneously in isolated segments of paired R- and L-MCAs, testing responses to electrical field stimulation and the presence of the alpha-adrenoceptor antagonist, phentolamine, at different concentrations and times post-SAH. Fractional 3H-NA efflux from ruptured R-MCAs was reduced to undetectable levels at 18 h to at least 3 d post-SAH. Response to electrical stimulation partially recovered at 10 d and approached the controls by 16 to 30 d. In the L-MCAs, 3H-NA efflux was decreased up to 90% at 18 h, but recovered to control level by 3 d, unless it too became involved by encroachment of blood from the SAH side. The fractional 3H-NA efflux in the controls was typically augmented by phentolamine, reaching a peak at 0.3 microM of the drug. This overflow response was completely lost between 0.03 and 3.0 microM phentolamine in the R-MCAs for at least 30 d post-SAH, whereas uninvolved L-MCAs regained drug-induced overflow at 10 to 16 d post-SAH. Uptake of 3H-NA after SAH was also decreased 30 to 50% for both MCAs at 18 h and 3 d post-SAH. Control 3H-NA uptake was regained by 10 d post-SAH in the L-MCA but not until 16 d in the R-MCA.(ABSTRACT TRUNCATED AT 250 WORDS)
Neurological Research 04/1988; 10(1):49-56. · 1.52 Impact Factor
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Pathology and Immunopathology Research 02/1987; 6(3):190-206.
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ABSTRACT: It has been hypothesized that chemical interactions between neurons in the central nervous system can occur in the absence of well defined synaptic complexes, but morphological correlates have been difficult to find. The present study demonstrates exocytotic release from large (70-130 nm) dense cored vesicles at structurally nonspecialized areas along the plasmalemma of structurally different categories of terminals and occasionally from dendrites and axons within the neuropil of the trigeminal subnucleus caudalis. In rats, the marginal (lamina I) and substantia gelatinosa (lamina II) layers contain the central terminals of primary afferent fibers from the infraorbital nerve that supply the skin and whiskers (vibrissae). Different types of interneurons are also present and may modify the input being relayed to higher centers. While exocytotic profiles were present in control animals, they increased significantly (P less than 0.01) on the ipsilateral side 1-24 h after a unilateral skin lesion in the vibrissae area. A second increase (P less than 0.001) occurred 14-15 days after the lesion. Virtually all examples of large vesicle exocytosis were observed at structurally nonspecialized sites while those at the active synaptic zones involved small clear vesicles. Substance P-like immunofluorescence, present in controls and on the ipsilateral side during the first 6 days, subsequently declined until 4 weeks after surgery when some recovery was noted. The increase in large vesicle exocytosis and the decrease in substance P are interpreted to reflect functional adjustments of different neurons in response to the lesion. The exocytosis involving large dense cored vesicles may serve to deliver transmitters and/or neuropeptide modulators to appropriate receptors in a wider area than release into a specialized synaptic cleft would allow.
Neuroscience 10/1986; 19(1):43-54. · 3.38 Impact Factor
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ABSTRACT: Leukotriene B4 (LTB4) is a derivative of arachidonic acid which causes neutrophil diapedesis, endothelial swelling and increased permeability of post-capillary venules. To detect whether these effects are accompanied by degranulation of leukocytes and visible injury to the microvessels, the vasculature of rabbit skeletal muscle (tenuissimus) was exposed to LTB4 (10-20 nM). Some preparations were pre-treated with prostaglandin E1, (PGE1). When leukocytes started to adhere markers of plasma leakage were infused. Ultrastructural examination of leakage areas revealed that neutrophils and eosinophils appeared structurally intact, but many basophils and mast cells had been partially degranulated which indicated that vasoactive substances may have been liberated. However, endothelial gaps, such as may form in response to histamine released during degranulation, were not observed and there was no obvious injury to the endothelial cells. The apparent swelling observed by light microscopy was due to pseudopods of migrating leukocytes. Electron dense markers occurred in some endothelial vesicles and in the vicinity of neutrophils which had reached the abluminal side. These particles are interpreted to have escaped concurrent with leukocyte migration. After treatment with both PGE1 and LTB4 a few post-capillary venules showed endothelial gaps. However, leakage of markers was insignificant where the basement membrane persisted. It is concluded that exposure to LTB4 per se and the resulting leukocyte diapedesis are not structurally damaging to the vasculature.
Tissue and Cell 02/1986; 18(1):1-12. · 1.04 Impact Factor
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ABSTRACT: Nerve terminals often contain morphologically distinct populations of large (75-110 nm) and small (45-55 nm) vesicles. The small vesicles are speculated to account for release of transmitter quanta as they accumulate at presynaptic membranes. Large vesicles can co-store neuropeptides and classical transmitters but their function in neurotransmission has been disputed because they do not appear to accumulate at chemical synapses. However, there is now evidence that the large vesicles play a role in neurotransmission or its modulation even though they may not be eminently involved in synaptic release. Thus, exocytosis occurs along the synapse-lacking membranes of peripheral noradrenergic varicosities. Large vesicles may continue to function in peptide release even after the classical transmitter has been depleted as demonstrated in the pig vas deferens. Three days of reserpine administration causes a parallel loss of noradrenaline and small vesicle contents but does not decrease enkephalin-like immunoreactivity or large vesicle electron density. In the central nervous system of the rat, where substance P and enkephalin have been localized to large vesicles, exocytosis occurs from several types of terminals. The large vesicles appear preferentially to release their contents at morphologically non-specialized sites even when characteristic synapses are present. Thus different mechanisms of transmitter and neuropeptide release may coexist. The nonsynaptic discharge may allow substances to diffuse over a wider distance whereas release into a synaptic cleft could restrict receptor interaction.
Scanning electron microscopy 02/1986;
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Acta Physiologica Scandinavica 11/1984; 122(2):221-4. · 2.55 Impact Factor
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ABSTRACT: Bovine splenic vein has an abundant sympathetic innervation. Isolated strips were used to examine whether autoinhibition of norepinephrine release from the noradrenergic nerve terminals could be demonstrated under various experimental conditions and whether additional local regulatory modulators of transmitter release could also be implicated. In particular, the possibility of a histamine interaction with presynaptic inhibitory receptors was examined because ultrastructural evidence disclosed a close spatial relationship between mast cells and noradrenergic nerve terminals in the vessel wall. To investigate the presence of presynaptic alpha-receptors the competitive blocking agent phentolamine was included in the superfusion medium at concentrations ranging from 1 to 50 microM during electrical field stimulation at frequencies between 1 and 10 Hz. Transmitter outflow was measured as fractional tritium release. Low frequency stimulation (1 Hz) with 1 microM phentolamine resulted in the typical increase in norepinephrine release characteristics for presynaptic alpha-receptor inhibition. In contrast, high frequency (10 Hz) stimulation in the presence of 50 microM phentolamine caused an unexpected decrease in norepinephrine outflow. This unusual result can be explained by additional pharmacological actions of phentolamine unrelated to alpha-receptor blockade, e.g. histamine release from the mast cells which subsequently can act on presynaptic inhibitory histamine receptors. This effect, manifested at higher phentolamine concentrations, would overcome the alpha-receptor blockade. The presence of histamine receptors was supported by the results from electrical stimulation in the presence of exogenous histamine. Histamine decreased norepinephrine outflow while increasing basal tension and the contractile response of the vein strip. Unexpectedly, these effects appeared to be mediated by histamine receptors of the H1-type because they were reduced after pyrilamine but unaffected by agonists and antagonists to receptors of the H2-type. It is speculated that interactions between mast cells and noradrenergic nerve terminals may serve to maintain homeostasis in the bovine splenic vein.
Blood vessels 02/1983; 20(3):122-34.
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ABSTRACT: The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.
Journal of Neurobiology 06/1979; 10(3):309-24. · 3.05 Impact Factor
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Neuroscience 02/1977; 2(4):621-34. · 3.38 Impact Factor
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ABSTRACT: Adult rats maintained at 20-22 degrees C, were exposed to 4 degrees C for 30-60 min and injected with 50 or 100 mg/kg 6-hydroxydopamine (6-OHDA) in an attempt to achieve a similar degree of chemical sympathectomy of nerves terminating among the adipocytes and the smooth muscle cells of the blood vessels in the interscapular brown adipose tissue (BAT). After 1, 4 or 10 days the pads of BAT were removed and small sections from each pad prepared for electron microscopy; the remaining tissue was used for noradrenaline (NA) analysis for fluorescence histochemistry. Ultrastructural observations showed that 24 h after the 6-OHDA injection virtually all noradrenergic nerve terminals were distorted and contained aggregates of degenerated cell organelles. The destruction could be correlated with a disappearance of fluorescent varicosities and a reduction of measurable NA to 8-12% of the control value. There was no differential toxic effect of 6-OHDA on the terminals among the adipocytes compared to those associated with blood vessels. Thus, treatment with 6-OHDA is more effective than previous attempts using immunological or surgical methods to produce sympathectomy, because both of the latter methods only eliminate the innervation of the blood vessels and spare the nerve terminals of the adipocytes. 4 days after 6-OHDA injection there was no improvement in the morphology of the terminals but after 10 days there was an increase in the number of terminals and axons with a normal appearance and this was paralleled by an increase in extractable NA to 50% of the control value. Because of the relatively rapid recovery of NA content and reappearance of terminals of normal appearance within 10 days after 6-OHDA injection, these animals should be injected weekly when a more permanent sympathectomy of adipocytes and blood vessels is desired.
Journal of Neurocytology 01/1977; 5(6):677-90. · 1.94 Impact Factor
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ABSTRACT: Adult non-cold adapted rats were injected with 6-hydroxydopamine (6-OHDA) or saline and their interscapular brown adipose tissue (BAT) was removed after appropriate periods of time. Fluorescence histochemistry of control BAT demonstrated the presence of an extensive network of varicose fibers among the adipocytes and at the blood vessels. This was confirmed by electron microscopy which also revealed large and small dense core vesicles sparsely distributed in axons and terminals indicating the presence of noradrenaline (NA). After 6-OHDA injection the fluorescence from varicosities was abolished both among the adipocytes and at the vessels. Thus, chemical sympathectomy was more effective than surgical- or immunosympathectomy, which spare the innervation of adipocytes. Parallelling the disappearance of fluorescence was a significant decrease of measurable NA. During recovery the extractable NA increased before the reappearance of fluorescent varicosities. This could be explained by transmitter accumulation in the nervetrunks within the tissue, which, in general, appeared unaffected by 6-OHDA. A large number of cells with a strong yellowish fluorescence distributed through the BAT was unaffected by 6-OHDA. There was no evidence for the presence of intrinsic ganglia.
Acta Physiologica Scandinavica 10/1976; 98(1):8-18. · 2.55 Impact Factor
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ABSTRACT: To study the ultrastructural characteristics of the sympathetic nerve terminals of human omental veins, and of their relationship to the innervated smooth muscle cells, biopsy specimens were taken during abdominal surgery, rapidly fixed in glutaraldehyde/osmium and stained with uranylacetate. The results indicate that the veins have an extensive noradrenergic innervation, penetrating into the tunica media. The distance between nerve terminals partly or wholly free from enveloping Schwann cells, to the surface of smooth muscle cells ranged from 30 to 500 nm. Large dense core vesicles were prominent in both preterminals and terminal regions, while small dense core vesicles occurred mainly in terminals. Large dense core vesicles in close contact with the axolemma were occasionally observed, indicating involvement in secretion by exocytosis.
Neuroscience 09/1976; 1(4):333-7. · 3.38 Impact Factor
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ABSTRACT: Norepinephrine (NE):adenosinetriphosphate (ATP) ratios were studied in a highly purified fraction of large dense core vesicles isolated from the bovine splenic nerve. Vesicles prepared from nerves chilled approximately 10 and 30 min post mortem were compared. The NE:ATP molar ratio decreased from 6.3 to 4.8, p less than 0.005; NE decreased from 61 to 42 nmol, while ATP decreased only from 9.6 to 8.8 nmol/mg protein. Animals weighing 180-360 kg were compared with heavier ones weighing 400-700 kg. NE increased from 42 to 68 nmol and ATP increased from 5.9 to 13.2 nmol/mg protein, while the NE:ATP molar ratio decreased from 7.2 to 5.2, p less than 0.005. Changes during vesicle maturation were studied by comparing vesicles identically prepared from equal weights of a proximal nerve segment close to the coeliac ganglion and a distal, intrasplenic segment. NE increased from 45 to 70 nmol while ATP remained unchanged at 10.0 nmol/mg protein and the NE:ATP molar ratio increased from 4.5 to 7.0, p less than 0.005. It was interpreted that vesicle ATP content, like dopamine beta-hydroxylase, was established early in the cell body and remained unchanged during axoplasmic transport. ATP was in a complex which was relatively stable to post mortem hydrolysis at least between 10 and 30 min prior to chilling the nerves. The addition of newly synthesized NE into a readily releasable pool during axoplasmic transport occurs without ATP and can account for the increased ratio above 4:1 in the distal segment vesicles.
Journal of Neurobiology 02/1976; 7(1):11-22. · 3.05 Impact Factor
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ABSTRACT: Large dense core vesicles (LDV) were purified from bovine splenic nerve homogenates by the sucrose-D2O density gradient method. Vesicles were subjected to a 50% increase and decrease in osmolarity from the control 330 mosmol 1(-1) by adjusting sucrose or potassium phosphate buffer during pre- and/or postfixation. Control vesicles with a mean diameter of 717 A readily swelled to approximately 1050 A and shrunk to approximately 600 A in the hypotonic and hypertonic media, respectively, with either sucrose or phosphate buffer. The dense core responded similarly but to a lesser degree. Prefixation in glutaraldehyde had little effect on vesicle sensitivity to subsequent tonicity change, not did the fixative per se exert an obvious osmotic effect. Thus, final vesicle size was largely determined by the OsO4 postfixation medium and principally by the vehicle rather than the fixative. In controls there was a mixture of spherical to oblate vesicles mostly filled with an electron-dense matrix. Upon swelling, more vesicles became spherical and nearly all had a prominent translucent halo between core and membrane. Upon shrinking, more vesicles became oblate, the halo was obliterated and the electron-density of the matrix increased. Frequency distributions of vesicle diameters at different tonicity clearly indicated that the diameter of LDV could overlap the 400-500 A range characteristic of small dense core vesicles; however, there was no suggestion of a population of the latter in the purified LDV fraction. Implications are discussed concerning the biochemical and morphological identification of 'light' and 'heavy' density peaks of noradrenaline and dopamine beta-hydroxylase from mixed vesicle populations and the possible relevance of changes in vesicle shape to a functional state in situ.
Journal of Neurocytology 11/1975; 4(5):609-27. · 1.94 Impact Factor
