Biomedical subjects

S M Strittmatter

Publications and source records attributed to S M Strittmatter.

70 records · Page 4Linked to original sources

PubMed1986

Enkephalin convertase: localization to specific neuronal pathways.

3H-Guanidinoethylmercaptosuccinic acid (GEMSA) selectively labels the carboxypeptidase B-like enzyme enkephalin convertase (EC) in rat brain tissue sections. We have used autoradiography with 3H-GEMSA to map membrane-bound EC in the rat forebrain and, in conjunction with lesioning techniques, to localize EC to specific neuronal pathways. The highest levels of EC are in the median eminence. High levels of EC also occur in the hypothalamic magnocellular nuclei, in several nuclei of the amygdala, the lateral septum, and the bed nuclei of the stria terminalis. Knife-cut lesions of the stria terminalis increase EC posterior to the lesion in the stria and deplete EC from the stria adjacent to the bed nucleus, suggesting that EC, like enkephalins, is axonally transported within the stria terminalis. Ibotenic acid lesions of the caudate nucleus destroy binding in the substantia nigra pars reticulata ipsilateral to the lesion, suggesting that nigral EC is associated with axons originating in the caudate nucleus. We have also mapped EC in detail in the hippocampus. EC levels are highest near pyramidal cells of CA 3-4 and the dentate gyrus granule cells. Quinolinic acid lesions destroy both the granule and pyramidal cells and destroy all of the 3H-GEMSA labeling except for a small amount in the molecular layer of the dentate gyrus. Selective destruction of CA 3-4 pyramidal cells with kainic acid eliminates EC in the pyramidal cell region. Destruction of granule cells of the dentate gyrus with colchicine depletes binding in the dentate gyrus without any change in the area surrounding field CA 3-4. High levels of 3H-GEMSA binding are present in the hippocampus at least 3 d before birth. These observations suggest that in the hippocampus the majority of EC is associated with pyramidal cells, which have not been shown to contain enkephalins. 3H-GEMSA autoradiography of the trigeminal ganglion localizes EC to the sensory neurons and not to white matter tracts there. These studies demonstrate that while EC is contained in enkephalinergic pathways, it is also present in some neurons that do not contain enkephalins.

Animals↗

PubMed1985

A rat brain isozyme of angiotensin-converting enzyme. Unique specificity for amidated peptide substrates.

We have purified angiotensin-converting enzyme (ACE, EC 3.4.15.1) from rat brain corpus striatum and rat lung. The brain enzyme has Mr 165,000 by sodium dodecyl sulfate gel electrophoresis, whereas the lung enzyme is 175,000. This difference is not an artifact of preparation since mixture of the two tissues prior to purification results in isolation of two proteins with Mr 165,000 and 175,000. Separation of tryptic fragments of 125I-labeled lung and brain ACE by reverse-phase chromatography yields distinct but similar patterns. No differences between the native enzymes are detected in dansyl-tripeptide cleavage specificity, inhibitor profile, immunological properties, sucrose gradient sedimentation, or gel filtration of ACE from the two tissues. However, lung and brain ACE can be differentiated in their ability to cleave amidated peptides. Both lung and brain ACE cleave Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) via two pathways. In one pathway, ACE first releases Gly-Leu-Met-NH2 and then dipeptides sequentially from the carboxyl terminus. The other first produces Leu-Met-NH2, and then releases dipeptides to leave substance P 1-5. Lung ACE favors initial tripeptide release 3:1, while the striatal enzyme acts via the two pathways to a similar extent. Lung and striatal ACE also differ in their ability to degrade other amidated peptides. His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (substance K) and bombesin are degraded by striatal but not lung ACE. Physalaemin and luteinizing hormone-releasing hormone are cleaved by both enzymes, while eledoisin, kassinin, thyrotropin-releasing hormone, and substance P 5-11 are not cleaved by either enzyme. Physalaemin is degraded more rapidly by the lung enzyme. The coincidence of an ACE isozyme with substance P and substance K in the descending striatonigral pathway and the unique ability of this isozyme to cleave substance P and substance K suggest that one or both of these peptides is a physiological substrate for striatonigral ACE.

Amino Acid Sequence↗

PubMed1985

Substance K and substance P as possible endogenous substrates of angiotensin converting enzyme in the brain.

In the brain angiotensin converting enzyme is highly localized to a striatonigral pathway, which contains no endogenous angiotensin. Substance P, also localized to a striatonigral pathway, is degraded by ACE via two different pathways. The lung and striatal isozymes of angiotensin converting enzyme exhibit differential cleavage of substance P, with lung preferring an initial tripeptide cleavage, and striatum an initial dipeptide cleavage. Substance K is degraded by the striatal isozyme but is not cleaved by the lung isozyme. Substance P 5-11 is not cleaved by either form of angiotensin converting enzyme.

Amino Acid Sequence↗

PubMed1985

Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity.

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathological and clinical abnormalities in humans, monkeys, and mice that closely resemble idiopathic parkinsonism. N-Methyl-4-phenylpyridine (MPP+), a metabolite of MPTP formed by monoamine oxidase B, is accumulated into striatal and cerebral cortical synaptosomes by the dopamine and norepinephrine uptake systems, respectively, whereas MPTP itself is not accumulated. The potencies of drugs in inhibiting [3H]MPP+ or [3H]dopamine uptake into striatal synaptosomes are very similar, as are potencies in inhibiting [3H]MPP+ or [3H]norepinephrine uptake into cortical synaptosomes. The Km values for [3H]MPP+ uptake are 170 and 65 nM and the Vmax values are 2 and 0.1 nmol/g of tissue per min in rat striatum and cortex, respectively, similar to values for [3H]dopamine uptake, Autoradiography of accumulated [3H]MPP+ in slices of rat brain shows high densities in the caudate-putamen and nucleus accumbens. Furthermore, blockade of dopamine uptake by mazindol prevents MPTP-induced damage to nigrostriatal dopamine neurons, indicating that MPP+ concentration into dopamine neurons explains their selective destruction by MPTP.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1985

Isolation and characterization of an olfactory receptor protein for odorant pyrazines.

The highly potent bell pepper odorant 2-isobutyl-3-[3H]methoxypyrazine [( 3H]IBMP) binds specifically and saturably to bovine and rat nasal epithelium. Specific binding is not detected in 11 other tissues assayed, and in the rat binding is 9 times higher in olfactory than in respiratory epithelium. We have purified to apparent homogeneity a soluble pyrazine odorant binding protein that constitutes approximately equal to 1% of the total soluble protein in bovine nasal epithelium. Polyacrylamide gel electrophoresis shows a single band of 19,000 Da and gel filtration data suggest that the native protein is a dimer of 38,000 Da. Binding of [3H]IBMP to the purified protein reveals two binding sites (Kd = 10 X 10(-9) M, Bmax = 135 pmol per mg of protein; Kd = 3 X 10(-6) M, Bmax = 25 nmol per mg of protein). The binding affinities of a homologous series of pyrazine odorants correlate with the human odor detection thresholds of these compounds. This correlation, together with the regional distribution of the protein, suggests that the protein is a physiologically relevant olfactory receptor.

Animals↗

PubMed1985

Angiotensin-converting enzyme in the testis and epididymis: differential development and pituitary regulation of isozymes.

Angiotensin-converting enzyme (ACE, EC 3.14.5.1) is found in particulate fractions of the epididymis but not in soluble epididymal fractions or in the testis of 4-week-old rats. [3H]Captopril autoradiography of testis and epididymis from 4-week-old rats confirms the association of ACE with epididymal ducts but not the testis. ACE appears in the testis between 4 and 6 weeks of age. Soluble ACE is not detectable in the epididymis until 6-7 weeks of age. Within the epididymis, regions closest to the testis develop soluble ACE activity about 1 week before those nearest to the vas deferens. Hypophysectomy of 10 week-old-rats depletes greater than 95% of ACE activity from the testis and soluble fractions of the epididymis, with little change in ACE levels from particulate fractions of the epididymis. [3H]Captopril autoradiography after hypophysectomy reveals luminal and epithelial ACE in the epididymis. The presence of particulate ACE in the epididymis under conditions where there is no testicular ACE indicates that the two forms are synthesized separately. However, soluble ACE from the epididymis might be derived from the membrane-associated ACE of the testis. Such a relationship is supported by the lag of 1 week between the development of ACE in the initial segment of the epididymis and the tail of the epididymis, and by the occurrence of soluble epididymis ACE only in those animals with testicular ACE activity.

Animals↗

PubMed1985

Enkephalin convertase demonstrated in the pituitary and adrenal gland by [3H]guanidinoethylmercaptosuccinic acid autoradiography: dehydration decreases neurohypophyseal levels.

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography demonstrates the particulate form of a carboxypeptidase B-like peptide processing enzyme, enkephalin convertase (EC 3.4.17.10), in the rat pituitary and adrenal glands. The maximal number of binding sites (Bmax) for [3H]GEMSA is 20 pmol/mg protein in the intermediate lobe of the pituitary, 12.0 pmol/mg protein in the posterior pituitary lobe, 15 pmol/mg protein in the anterior pituitary lobe, 5.8 pmol/mg protein in the adrenal medulla, and less than 0.3 pmol/mg protein in the adrenal cortex. The labeling pattern is homogeneous within each of these regions. Subcellular fractionation of the bovine adrenal medulla demonstrates that [3H] GEMSA-binding sites are localized to chromaffin granules. In Brattleboro rats and dehydrated rats, the level of posterior pituitary [3H]GEMSA binding is less than 25% of that in control animals. This decrease is abolished by arginine vasopressin treatment of Brattleboro rats or rehydration of dehydrated rats. There are no changes in [3H] GEMSA binding in the supraoptic nucleus or magnocellular portion of the paraventricular nucleus of the hypothalamus under any of these conditions, suggesting that the alterations observed in the neurohypophysis result from an increased rate of loss of enkephalin convertase. The level of anterior pituitary enkephalin convertase is unchanged by dehydration, adrenalectomy, or dexamethasone or in Brattleboro rats. [3H]GEMSA labeling in the intermediate pituitary lobe is unaffected by dehydration and haloperidol treatment and in Brattleboro rats. The adrenal medullary enzyme is not altered by reserpine, hypophysectomy, or splanchnic denervation or in Brattleboro rats.

Adrenal Glands↗

PubMed1985

Differential visualization of dopamine and norepinephrine uptake sites in rat brain using [3H]mazindol autoradiography.

Mazindol is a potent inhibitor of neuronal dopamine (DA) and norepinephrine (NE) uptake. DA and NE uptake sites in rat brain have been differentially visualized using [3H]mazindol autoradiography. At appropriate concentrations, desipramine (DMI) selectively inhibits [3H]mazindol binding to NE uptake sites without significantly affecting binding to DA uptake sites. The localization of DMI-insensitive specific [3H] mazindol binding, reflecting DA uptake sites, is densest in the caudate-putamen, the nucleus accumbens, the olfactory tubercle, the subthalamic nucleus, the ventral tegmental area, the substantia nigra (SN) pars compacta, and the anterior olfactory nuclei. In contrast, the localization of DMI-sensitive specific [3H]mazindol binding, representing NE uptake sites, is densest in the locus coeruleus, the nucleus of the solitary tract, the bed nucleus of the stria terminalis, the paraventricular and periventricular nuclei of the hypothalamus, and the anteroventral thalamus. The distribution of DMI-insensitive specific [3H]mazindol binding closely parallels that of dopaminergic terminal and somatodendritic regions, while the distribution of DMI-sensitive specific [3H]mazindol binding correlates well with the regional localization of noradrenergic terminals and cell bodies. Injection of 6-hydroxydopamine, ibotenic acid, or colchicine into the SN decreases [3H]mazindol binding to DA uptake sites in the ipsilateral caudate-putamen by 85%. In contrast, ibotenic acid lesions of the caudate-putamen do not reduce [3H]mazindol binding to either the ipsilateral or contralateral caudate-putamen. Thus, the DA uptake sites in the caudate-putamen are located on the presynaptic terminals of dopaminergic axons originating from the SN.

Animals↗

PubMed1984

[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase.

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.

Animals↗

PubMed1984

A fluorometric assay for angiotensin-converting enzyme activity.

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.

Animals↗

PubMed1984

Enkephalin convertase localization by [3H]guanidinoethylmercaptosuccinic acid autoradiography: selective association with enkephalin-containing neurons.

Enkephalin convertase, an enkephalin-forming carboxypeptidase, is potently inhibited by guanidinoethylmercaptosuccinic acid (GEMSA). We have localized enkephalin convertase in rat brain by in vitro autoradiography with [3H]GEMSA. [3H]GEMSA-associated silver grains are highly concentrated in the median eminence, bed nucleus of the stria terminalis, lateral septum, dentate gyrus, hippocampus, central nucleus of the amygdala, preoptic hypothalamus, magnocellular nuclei of the hypothalamus, interpeduncular nucleus, dorsal parabrachial nucleus, locus coeruleus, nucleus of the solitary tract, and the substantia gelatinosa of the spinal trigeminal tract. This distribution corresponds closely with immunocytochemical localizations of enkephalin-containing cells and axons, indicating that enkephalin convertase is selectively involved in enkephalin biosynthesis.

Animals↗

PubMed1984

Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: localization to a striatonigral pathway.

We have visualized angiotensin-converting enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) in rat brain by in vitro [3H]captopril autoradiography. [3H]Captopril binding to brain slices displays a high affinity (Kd = 1.8 X 10(-9) M) and a pharmacological profile similar to that of ACE activity. Very high densities of [3H]captopril binding were found in the choroid plexus and the subfornical organ. High densities were present in the caudate putamen and substantia nigra, zona reticulata. Moderate levels were found in the entopeduncular nucleus, globus pallidus, and median eminence of the hypothalamus. Lower levels were detectable in the supraoptic and paraventricular nuclei of the hypothalamus, the medial habenula, the median preoptic area, and the locus coeruleus. Injection of ibotenic acid or colchicine into the caudate putamen decreased [3H]captopril-associated autoradiographic grains by 85% in the ipsilateral caudate putamen and by greater than 50% in the ipsilateral substantia nigra. Thus, ACE in the substantia nigra is located on presynaptic terminals of axons originating from the caudate putamen, and ACE in the caudate putamen is situated in neuronal perikarya or at the terminals of striatal interneurons. The lack of effect of similar injections into the substantia nigra confirmed that the caudate putamen injections did not cause trans-synaptic changes. The presence of [3H]captopril binding is consistent with an ACE-mediated production of angiotensin II in some brain regions. Although [3H]captopril autoradiography reveals ACE in a striatonigral pathway, there is no evidence for angiotensin II involvement in such a neuronal pathway.

Animals↗

PubMed1984

Angiotensin-converting enzyme in the male rat reproductive system: autoradiographic visualization with [3H]captopril.

[3H]Captopril autoradiography visualizes angiotensin-converting enzyme (ACE; EC 3.14.5.1) in the reproductive tract of male rats. [3H]Captopril binds to testicular slices with high affinity (Kd = 4.4 nM) and displays a pharmacological profile similar to that of ACE activity. High densities of [3H] captopril autoradiographic silver grains are found over spermatid heads and in the lumen of seminiferous tubules in stages I-VIII and XII-XIV. Tubules in stages IX-XI exhibit only one fifth the level of binding. The basal epithelium and interstitial tissue are not labeled. The initial segment of the epididymis contains very low levels of grains. The head of the epididymis demonstrates intense grain density at the luminal surface of the epithelium, with little luminal labeling. In a progression to the tail of the epididymis, epithelium labeling declines, and luminal grains increase. The lumen of the vas deferens is also labeled. ACE from testis and several regions of the epididymis has been categorized with respect to its particulate vs. soluble nature and its ability to be precipitated by an antirat lung ACE monoclonal antibody. Testicular ACE is particulate and not immunoprecipitable. The distribution of immunoprecipitable particulate ACE in the epididymis is similar to that of autoradiographic silver grains over the epithelium. The concentration of particulate but nonprecipitable ACE gradually rises from the initial segment to the tail of the epididymis, similar to the distribution of luminal [3H]captopril-associated grains. Soluble ACE activity, present in equal concentration throughout the epididymis and not immunoprecipitable, may not be detected by autoradiography of [3H]captopril.

Animals↗

PubMed1983

[3H]Captopril binding to membrane associated angiotensin converting enzyme.

[3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a KD of 2.4 nM. [3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [3H]captopril binds selectively to angiotensin converting enzyme. [3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.

Animals↗

PubMed1982

Physical separation and characterization of two types of benzodiazepine receptors.

Two distinct benzodiazepine receptors are solubilized differentially by various detergents. The receptor sites that resist solubilization, designated type I, are most highly concentrated in the cerebellum and corpus striatum whereas the more readily solubilized receptors, type II, are most enriched in the hippocampus. The type I receptors display higher affinity for beta-carboline esters and a triazolopyridazine whereas several benzodiazepines do not differentiate the two receptors. The type I receptors can be solubilized with 2% Triton X-100/1 M NaCl; they retain the same drug specificity as in the particulate state.

Animals↗

PubMedDate not supplied

Monoclonal antibody production by receptor-mediated electrically induced cell fusion.

Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

Animals↗