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S E Leeman

Publications and source records attributed to S E Leeman.

At least 73 records · Page 4Linked to original sources
PubMed1988

Synaptic vesicles from mammalian brain: large-scale purification and physical and immunochemical characterization.

Purification of synaptic vesicles directly from homogenates of mammalian brain is compared with a classical method based on osmotic lysis of brain synaptosomes. The direct method affords increased yield and purity of synaptic vesicles prepared under isoosmotic conditions. Antigen SV2 and the antigens (primarily synaptophysin) recognized by rabbit antiserum R10, raised to purified rat brain synaptic vesicles, are localized specifically on approximately 40-nm-diameter microsomal vesicles from rat brain. Rat brain synaptic vesicles have equilibrium densities of approximately 1.11 g/ml on Nycodenz density gradients, 1.12 g/ml on glycerol/Nycodenz, and 1.07 g/ml on Ficoll gradients. Both SV2 and the R10 antigens are enriched approximately 50-fold in purified rat brain synaptic vesicles. Synaptic vesicles purified from rat or cow brain show active uptake of [3H]norepinephrine that is reserpine sensitive and dependent on ATP and Mg2+. Synaptic vesicles exhibiting [3H]norepinephrine uptake comigrate with approximately 40-nm-diameter synaptic vesicles carrying SV2 or R10 antigens during permeation chromatography. After the Sephacryl S-1000 chromatography step, [3H]-norepinephrine uptake activity is purified approximately 90-fold. Highly purified brain synaptic vesicles should facilitate studies at the molecular level of the roles of these organelles in neurotransmission at mammalian synapses.

Animals↗
PubMed1988

Identification and characterization of the major proteins of mammalian brain synaptic vesicles.

Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of approximately 74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle-specific monoclonal antibody. The 40,000-Mr protein appeared to be identical to the 38,000-Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000-Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000-Mr protein is approximately 5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these proteins.

Animals↗
PubMed1987

Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells.

1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time- and concentration-dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non-responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first-order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist-mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist-mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM-carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM-SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half-maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6-fold in the presence of 0.3 microM-SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist-mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist-mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary.

Substance P (SP) and substance K (SK) are mammalian tachykinin peptides derived from a single preprotachykinin-A (PPT-A) gene and are widely but selectively distributed in neural and endocrine tissues. SP is present in the rat anterior pituitary, and its content there varies with the thyroid status of the animal. The present studies were undertaken to determine whether the PPT-A gene is expressed in the anterior pituitary and if so, whether PPT-A messenger RNA (mRNA) abundance is regulated by thyroid hormone status. Male rats were surgically or chemically thyroidectomized or made hyperthyroid by thyroid hormone (T3) injection. Total RNA was isolated from individual anterior pituitaries, and PPT-A mRNA abundance was determined by dot blot procedures. In parallel groups of rats, anterior pituitaries were extracted for measurement of SP and SK by specific RIAs. Surgical or chemical thyroidectomy increased PPT-A mRNA abundance 4 to 6-fold and increased both SP and SK content in the anterior pituitary. Administration of T3 to thyroidectomized rats reversed the increase in both PPT-A mRNA abundance and SP and SK content in the adenohypophysis. T3 administration to euthyroid rats also decreased PPT-A mRNA abundance and SP and SK content in the anterior pituitary. The coordinate presence of PPT-A mRNA with SP and SK in the anterior pituitary strongly suggests that these peptides are synthesized within this gland.

Animals↗
PubMed1987

Diminished flare response in neuropathic diabetic patients. Comparison of effects of substance P, histamine, and capsaicin.

The flare response in skin largely depends on an intact primary sensory fiber, the C-fiber. We measured the flare response to the intradermal injection of substance P, histamine, and capsaicin in control subjects and in diabetic patients with and without clinically obvious polyneuropathy. The neuropathic diabetic patients had a reduced flare response to substance P, histamine, and capsaicin, compared with control and nonneuropathic diabetic subjects. The smaller flare response in the neuropathic diabetics after capsaicin administration suggested a dysfunction of the peripheral component of the C-fiber. Alternatively, dysfunction of the mast cell or vascular reactivity may contribute to the diminished flare. Because C-fibers participate in nociception in addition to the flare response, the findings of this study, by a method that permits a quantifiable measurement of the function of peripheral sensory neurons in diabetic subjects, has potential usefulness in evaluating sensory neuropathy in diabetic patients.

Capsaicin↗
PubMed1987

Desensitization to substance P-induced vasodilation in vitro is not shared by endogenous tachykinin neurokinin A.

Two mammalian tachykinins, substance P (SP) and neurokinin A (NKA), were measured by radioimmunoassay in canine cephalic blood vessels and tested for their vasoactivity in vitro. Levels of immunoreactive SP were approximately 2-3 times greater than those of immunoreactive NKA in common carotid, basilar, and middle cerebral arteries. Both endogenous tachykinins relaxed precontracted segments of common carotid and basilar arteries in a dose-dependent manner with an EC50 of 8.9 X 10(-11) M and 7 X 10(-10) M, respectively, when added cumulatively. Relaxation was endothelial dependent for both substances and not blocked or enhanced by pretreatment with indomethacin, propranolol, lithium chloride, or atropine. Neither SP nor NKA released 3H-inositol phosphates from phospholipid membranes of canine carotid segments after preincubation with 3H-inositol. SP but not NKA or the C-terminal fragments SP(4-11) caused desensitization to subsequent additions of itself but not to the relaxation induced by sodium nitroprusside, calcitonin gene-related peptide, or bradykinin. These studies demonstrate that at least 2 peptides derived from beta-preprotachykinin are contained within cephalic blood vessels and that these products share similar vasoactive properties but differ in their ability to desensitize vascular tachykinin receptors.

Animals↗
PubMed1986

Localization of aromatase and 5 alpha-reductase to neuronal and non-neuronal cells in the fetal rat hypothalamus.

Experiments were designed to identify the neural cell type(s) responsible for the aromatization and 5 alpha-reduction of androgens in the rat hypothalamus. Primary cultures of fetal rat hypothalamic cells, which had enhanced neuronal morphology, were treated at various times after plating with kainic acid (KA), a neurotoxic agent which selectively destroys neuronal cells. Neuronal morphology was disrupted in a time (0-6 days)- and dose (10(-4)-10(-2) M)-dependent fashion after KA treatment, with no apparent change in the appearance of the flattened, underlying non-neuronal cells. KA treatment for 4 days decreased aromatization by 94% in a dose-dependent fashion (10(-4)-10(-2) M KA), while 5 alpha-reduction declined by no more than 25%. A 6-day time course with 10(-3) M KA showed a dramatic decline in aromatization and no alteration in 5 alpha-reduction. In control experiments, substance P, a neuronal peptide, declined after KA treatment while the activity of glutamine synthetase, a glial enzyme, did not change. We conclude from these results that aromatase is localized primarily to neuronal cells in the hypothalamus while 5 alpha-reductase is confined primarily to non-neuronal cells.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1986

Multiple calcium channels mediate neurotransmitter release from peripheral neurons.

We examined the effects of dihydropyridine drugs on evoked neurotransmitter release from cultured neonatal rat sensory and sympathetic neurons. Depolarization with K+-rich solutions increased the release of substance P from cultured sensory neurons. This release was enhanced by BAY K8644 and (+)-202791 and was blocked by a variety of other dihydropyridines including (-)-202791, by Co2+, or in Ca2+-free solutions. K+-rich solutions also stimulated the release of [3H]norepinephrine from cultured sympathetic neurons. This release was also completely blocked by Co2+ or in Ca2+-free solution. In contrast to the situation in sensory neurons, however, the evoked release of [3H]norepinephrine was completely resistant to the blocking effects of dihydropyridine such as nimodipine. However, BAY K8644 was able to enhance the evoked release of [3H]norepinephrine, and this enhancement was blocked by nimodipine. These results are discussed in relation to the possible participation of multiple types of calcium channels in the release of neurotransmitters.

Animals↗
PubMed1986

Neurotensin stimulates [3H]oleic acid translocation across rat small intestine.

The effect of neurotensin (NT) on the translocation of intraluminally administered lipid across the duodenum as well as across the entire length of the small intestine was studied in the rat. In the first series of experiments, the appearance in the lymph of [3H]oleic acid instilled as a bolus into a segment of the duodenum was followed for 3 h. Infusion of NT (0.6 pmol X kg-1 X min-1) via the superior mesenteric artery resulted in a significant increase in the appearance of label in the lymph when compared with saline infusion (17.3 +/- 3.1 vs. 8.6 +/- 1.4%, P less than 0.01, respectively). In the second series of experiments, lipid was infused into the entire length of the small intestine over 4 h, and the accumulation of label in the lymph was measured. The infusion of NT (1.0 pmol X kg-1 X min-1) into the femoral vein also significantly increased the appearance of label when compared with animals infused with saline (49.0 +/- 2.0 vs. 34.2 +/- 5.2%, P less than 0.05, respectively). In this study, the specific activity of the triglyceride recovered in the lymph was higher in the rats given NT than in the controls (P less than 0.05). No significant changes in lymph flow were observed as a consequence of NT infusion. These results indicate that NT infusion into the circulation increases the translocation of oleic acid from the intestinal lumen into the lymph of rats.

Animals↗
PubMed1986

Chromatographic and immunochemical characterization of neurotensin in cat adrenal gland and its release during splanchnic nerve stimulation.

A subpopulation of norepinephrine-containing cells in the cat adrenal medulla contain neurotensin (NT) immunoreactive material. Using high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera we have demonstrated that electrical stimulation of the splanchnic nerve releases NT and its metabolites NT(1-8) and NT(1-12) from cat adrenal gland into the circulation. Blood samples from anesthetized cats were simultaneously collected from the adrenolumbar vein and femoral artery over a 10-min period prior to and during splanchnic nerve stimulation. Trains of stimuli (15 Hz/10V) were applied at 30-s intervals over 10 min. Plasma samples were extracted, run on HPLC and column fractions assayed with C- und N-terminal directed antisera. During splanchnic nerve stimulation unchanged. Concomitant with the increase in NT was a two- to threefold increase in the levels of NT(1-8) and NT(1-12). The chromatographic profiles of extracted adrenal glands showed a major peak of immunoreactive material with the same retention time as NT (18.6 +/- 3.0 pmol/g weight wet of tissue) as well as a small peaks on NT(1-8) (0.96 +/- 0.18 pmol/g) and NT(1-12) (2.05 +/- 0.1 pmol/g). HPLC analysis of a tryptic digest of the NT-like material gave 1.0 equivalent of NT(1-8) and NT(9-13). These results are consistent with the presence of NT in adrenal tissue and its release and metabolism during stimulation of the splanchnic nerve.

Adrenal Glands↗
PubMed1985

Evidence that large synaptic vesicles containing substance P and small synaptic vesicles have a surface antigen in common in rat.

Synaptic vesicles were purified from rat brain synaptosomes by osmotic lysis and chromatography on CPG-3000 controlled-pore glass beads. Large (approximately 100 nm) synaptic vesicles containing the peptide substance P (SP) were shown to be immunoprecipitated by a monoclonal antibody previously shown by W.D. Matthew, L. Tsavaler and L.F. Reichardt (J. Cell Biol., 91 (1981) 257-269) to selectively immunoprecipitate small (approximately 50 nm) synaptic vesicles from brain. Precipitation of SP-containing vesicles showed a saturable dependence on antibody concentration. These findings constitute direct, immunochemical evidence that large, peptidergic synaptic vesicles and small synaptic vesicles from brain have a surface antigen in common.

Animals↗
PubMed1985

Inhibition of flank-marking behavior in golden hamsters by microinjection of a vasopressin antagonist into the hypothalamus.

Microinjection of arginine-vasopressin (AVP) into the medial preoptic area of the hypothalamus of the hamster stimulates flank marking, a complex stereotypic motor behavior involved in olfactory communication. Microinjection of an antagonist of AVP, [1-deaminopenicillamine-2-(O-methyl)-tyrosine]arginine-vasopressin, into the same site blocks both the effect of microinjected AVP as well as the natural flank-marking behavior normally elicited by placing a hamster into the recently vacated home cage of another hamster. This finding supports the notion that AVP is a transmitter in the expression of flank marking.

Animal Communication↗
PubMed1985

Release and degradation of neurotensin during perfusion of rat small intestine with lipid.

The levels of neurotensin (NT) and its metabolite, the N-terminal octapeptide (NT1-8), identified by HPLC and measured by RIA, were increased in the hepatic-portal circulation of the anesthetized rat during perfusion of the small intestine with a lipid solution, while levels of both peptides remained unchanged in the general circulation. There was no significant arteriovenous difference for NT or NT1-8 during saline perfusion of the small intestine. Plasma collected from the superior mesenteric vein during the infusion of [3H]NT into the superior mesenteric artery showed major peaks of radioactivity with the retention times of NT1-8 and NT1-11 on HPLC. Only 12% of the radioactivity recovered from plasma was intact NT. These studies demonstrate that chromatographically identified NT and its metabolite, NT1-8, are elevated in the portal circulation but not systemic circulation during lipid perfusion and that the small intestine may be both the site of release and metabolism of NT.

Animals↗
PubMed1985

Alcohol and fatty acid stimulation of neurotensin release from rat small intestine.

We have previously reported that neurotensin (NT) is released from the small intestine and elevated in the hepatic-portal circulation in response to the perfusion of the small intestine with a micellar solution of oleic acid. In order to determine the minimum acyl chain length and whether the presence of a carboxylic acid is necessary for the stimulation of NT release, the small intestine of anesthetized rats was perfused with test solutions of fatty acids of 2-, 4-, 8-, or 18-carbons or fatty alcohols of 2-, 4-, or 8-carbons at a concentration of 1 mM prepared in 2.4 mM taurodeoxycholate in 0.9% NaCl. Blood samples, collected from the superior mesenteric vein immediately before the start of the test perfusion and at 15-min intervals thereafter, were extracted immediately and radioimmunoassayed for NT-like immunoreactivity (NTLI) with a C-terminal-directed antiserum. Perfusions of fatty acids with 4 or more carbons and alcohols of 2 or more carbons resulted in a significant elevation (P less than 0.05) in plasma levels of NTLI above the values obtained before the onset of perfusion. Perfusions with ethanol resulted in a value of 4.3 +/- 0.03 mg/dl (SEM) in blood from the superior mesenteric vein while there was no increase in ethanol levels in the peripheral circulation. Perfusion with taurodeoxycholate and 0.9% NaCl alone had no significant effect on plasma levels of the NTLI. In order to characterize the chemical nature of the elevated NTLI, plasma samples from animals perfused with test solution were collected, extracted, pooled, and subjected to HPLC. NT and its N-terminal metabolite, NT(1-8), were quantitated. NT was defined as material having the same retention time as synthetic NT standard and having comparable measurements using N- and C-terminal-directed antisera. Perfusions of fatty acids of four or more carbons and alcohols of two or more carbons resulted in a 2- to 4-fold increase of both NT and NT(1-8) levels in plasma. It is particularly interesting that perfusion with ethanol (2-carbons) causes an elevation in plasma NT, because perfusion with acetic acid (2-carbons) does not increase NTLI. The fact that perfusion of ethanol is effective in releasing intestinal NT suggests that NT may mediate some of the biological effects observed after the consumption of alcohol.

Acetates↗
PubMed1985

The differential effects of thyroid and gonadal hormones on substance P content in the anterior pituitary of the prepubertal rat.

The effects of thyroid and gonadal status on the content of substance P in the anterior pituitary (AP-SP) were examined in prepubertal rats. A sex difference in AP-SP is evident by age 50 days [males, 287 +/- 35 fmol/mg protein (mean +/- SE); females, 103 +/- 17; P less than 0.05], and this difference becomes greater by 75 days (males, 543 +/- 54; females, 146 +/- 11.5; P less than 0.01). Hypothyroidism was induced in male and female pups by giving lactating dams 0.1% methimazole (wt/vol) in their drinking water after parturition. There was a marked and significant increase in AP-SP in 21-day-old hypothyroid compared to euthyroid control pups. Male pups were made thyrotoxic by daily treatment with T4 (10 micrograms/rat, sc) from age 8 to 15 days. AP-SP was 4 times lower in the thyrotoxic than in the euthyroid pups (P less than 0.001). Rats ovariectomized at age 22 days and killed on day 35 revealed no change in AP-SP, in contrast to the rise in AP-SP in the ovariectomized adult rat. Female pups were treated with dihydrotestosterone (DHT; 50 micrograms/day) or testosterone (50 micrograms/day) from age 8-20 days. Neither androgen induced a change in AP-SP. Female pups which received estradiol (E2; 0.5 micrograms/day) or testosterone (75 micrograms/day) from age 8-20 days also had no change in AP-SP. As opposed to the lack of effect of E2 and DHT on AP-SP in female rats younger than 22 days, E2 (1 microgram/100 g BW daily) caused a decrease and DHT (100 micrograms/100 g BW daily) caused an increase in AP-SP in female rats treated from 22-35 days of age [E2, 91 +/- 6.9; DHT, 226 +/- 31 (P less than 0.05 vs. control for both); control, 154 +/- 13]. We conclude that the responsiveness of AP-SP to alterations in thyroid status is present at the youngest age studied. In contrast, the responsiveness of AP-SP to changes in the levels of gonadal steroids is absent in the infantile period and requires a maturational process that becomes evident during the juvenile state of sexual development.

Androgens↗
PubMed1984

Ultrastructural localization of immunoreactive neurotensin in the monkey superficial dorsal horn.

Neurotensin, a tridecapeptide, has been proposed to have a role in sensory systems, especially those mediating pain. The light microscopic and ultrastructural localization of neurotensin immunoreactivity in neurons of the monkey spinal cord was studied with the aim of examining their synaptic interactions. At the light microscopic level, neurotensin-containing cells were located in laminae II and III and immunoreactive axons and terminals were found in laminae I, II, and III. Neurotensin-positive axons were mostly thin and unmyelinated and their boutons contained both clear and large granular vesicles. Boutons varied considerably in size (1-3 micron) and in their relative content of large granular vesicles, which appeared occasionally in presynaptic locations. In lamina I neurotensin-immunoreactive terminals formed synapses with cell bodies which varied both in size and subcellular features. Some large dendrites in lamina I were contacted by numerous neurotensin-positive axons and also unlabeled terminals. In lamina II boutons with neurotensin immunoreactivity formed synapses mostly with small unlabeled dendrites some of which contained vesicles. The present results together with recent anatomical and physiological findings suggest that spinal cord neurons which contain neurotensin synapse with cells in the superficial dorsal horn that receive either input from primary afferents conveying nociceptive information or form part of the spinothalamic tract, or both. The diversity observed both in the morphology of neurotensin-positive terminals and in their synaptic patterns may indicate that they arise from more than one type of dorsal horn cell.

Animals↗
PubMed1984

Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region.

The suprachiasmatic nucleus has been identified tentatively as a circadian pacemaker. To examine the functional role of peptides found within suprachiasmatic neurons, avian pancreatic polypeptide and vasopressin were microinjected into the suprachiasmatic region. Avian pancreatic polypeptide, but not vasopressin, shifted the phase of the wheelrunning rhythm as a function of the time of its injection within the circadian cycle. Avian pancreatic polypeptide or a similar peptide may be one component of the neurochemical processes underlying entrainment to the light-dark cycle.

Animals↗