John E. Pintar

Transiently catecholaminergic (TC) cells in the bowel of the fetal rat: Precursors of noncatecholaminergic enteric neurons☆

Experiments were done to study the fate of transient catecholaminergic (TC) cells that develop in the rodent gut during ontogeny. When they are first detected, at Day E11 in rats, TC cells are distributed along the vagal pathway, in advance of the descending fibers of the vagus nerves, and in the foregut. The early TC cells coexpress the immunoreactivities of several neural markers, including 150-kDa neurofilament protein, peripherin, microtubule associated protein (MAP) 5, and growth-associated protein (GAP)-43, with those of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH). All cells in the fetal rat bowel at Day E11 that express neural markers also express TH immunoreactivity. The primitive TC cells also express the immunoreactivities of neural cell adhesion molecule (N-CAM), neuropeptide Y (NPY), and nerve growth factor (NGF) receptor (and NGF receptor mRNA). By Day E12 TC cells are found along the vagal pathway and throughout the entire preumbilical bowel. At this age TC cells acquire additional characteristics, including MAP 2 and synaptophysin immunoreactivities and acetylcholinesterase activity, which indicate that they continue to mature as neurons. In addition, TC cells of the rat are immunostained at Day E12 by the NC-1 monoclonal antibody, which in rats labels multiple cell types including migrating cells of neural crest origin. Despite their neural properties, at least some TC cells divide and therefore are neural precursors and not terminally differentiated neurons. At Day E10 TH mRNA-containing cells were not detected by in situ hybridization; however, by Day E11 TH mRNA was detected in sympathetic ganglia and in scattered cells in the mesenchyme of the foregut and vagal pathway. At this age, the number of enteric and vagal cells containing TH mRNA is about 30% less than the number of cells containing TH immunoreactivity in adjacent sections. The ratio of TH mRNA-containing cells to TH-immunoreactive vagal and enteric cells is even less at Day E12, especially in more caudal regions of the preumbilical bowel. A similar decline in the ratio of TH mRNA-containing to TH-immunoreactive cells was not observed in sympathetic ganglia. After Day E12 TH mRNA cannot be detected in enteric or vagal cells by in situ hybridization; nevertheless, TH immunoreactivity continues to be present through Day E14. DBH, NPY, and NGF receptor immunoreactivities are expressed by TH-immunoreactive transitional cells in the fetal rat gut after TH mRNA is no longer detectable.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences in the Structure of A and B Forms of Human Monoamine Oxidase

Abstract: [3H]Pargyline‐labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). [3H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [3H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS‐PAGE of [3H]pargyline‐bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS‐solubilized, [3H]pargyline‐labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one‐dimensional peptide mapping in SDS gels, different patterns of [3H]pargyline‐labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.

Insulin-like growth factor I and II and insulin-like growth factor binding protein-2 RNAs are expressed in adjacent tissues within rat embryonic and fetal limbs

Since the rapid proliferation of cells in a directed manner is a necessary component of limb formation, the distribution of locally produced mitogenic molecules within the developing limb is of considerable interest. We have used in situ hybridization to localize transcripts for both insulin-like growth factor binding protein-2 (IGFBP-2) and its ligands, the insulin-like growth factors I and II (IGF-I and IGF-II), within limb buds of rat embryos 10-16 days after conception (equivalent to stages 1-12 of mouse limb morphogenesis, Wanek et al, 1989. J. Exp. Zool. 249, 41-49). The mRNA for IGFBP-2 is very abundant in an anterior-posterior strip of ectoderm along the distal edge of the limb bud (the progenitor of the apical ectodermal ridge or AER) from as early as limb stage 1 (Embryonic Day 10) and is much less abundant in the rest of the limb ectoderm. A high level of IGFBP-2 expression continues to characterize the AER following its definitive appearance (stage 3) and throughout its existence (until stage 7). This is a period of rapid outgrowth during which the rate of mesodermal cell division is highest in cells nearest to the AER. The AER is known to have mitogenic activity in vitro and to direct limb outgrowth in vivo, but, until recently, few putative molecular correlates of these activities have been detected. The transcripts for IGF-I and IGF-II are also present at high abundance in developing limbs, especially in mesodermally derived cells. IGF-I mRNA is abundant in presumptive limb mesoderm from the beginning of limb outgrowth (just before stage 1), but is very low or undetectable in much of the rest of the embryo, while IGF-II mRNA becomes very abundant in limb mesoderm at stage 2. The distribution in limbs of both IGF-I and IGF-II mRNA changes dramatically during outgrowth and differentiation, so that their expression characterizes complementary populations of cells by stage 11. Taken together, these data suggest that IGFs and the IGF binding proteins, which may modulate IGF action, contribute to limb outgrowth and patterning.

Specificity of antisera prepared against pure bovine MAO-B

Antisera have been prepared against purified bovine MAO-B that appear to react selectively with MAO-B and not MAO-A, Rabbit and mouse antisera indirectly immune precipitated [125I]bovine MAO-B using inactivated Staphylococcus aureus cells, and binding of antibodies to bovine and rat MAO-B did not inhibit enzyme activity. Two continuous rat cell lines, hepatoma line MH1C1 and glioma line C6, were used to elucidate the specificity of the antisera. MH1C1 cells, which express both MAO-A and MAO-B, showed immune-specific staining with rabbit antiserum, and staining was blocked with pure MAO-B. Further, MAO-B activity and [3H]pargyline-labeled MAO molecules could be immune precipitated from solubilized mitochondrial preparations of MH1C1 cells; and immune fixation of mitochondrial proteins following SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed staining of the MAO-B, but not of the MAO-A, flavin-containing subunit. In contrast, no immune-specific immunocytochemical staining was observed in C6 cells, which have only MAO-A activity; no MAO-A activity or [3H]pargyline-labeled MAO could be immune precipitated from solubilized mitochondrial preparations of these cells, and no stained bands were observed for mitochondrial proteins resolved by SDS-PAGE and processed for immune fixation. Further support for the selectivity of this antiserum for MAO-B comes from immunocytochemical staining of rat tissues which express varying amounts of MAO-A and MAO-B activities. Hypothalamus and liver, with high levels of MAO-A and MAO-B activities showed a large number of immunoreactive cells, whereas spleen, heart and superior cervical ganglia, with high MAO-A and low MAO-B activities showed only a few or no stained cells. Catecholamine neurons in the substantia nigra, thought to contain MAO-A, did not show immune-specific staining. Skeletal muscle cells with low MAO-A and MAO-B activities did not stain. These studies provide additional evidence that MAO-A and MAO-B are distinct molecules, differentially expressed in different cell types.

Monoamine oxidase (MAO) activity as a determinant in human neurophysiology.

Several lines of evidence indicate that monoamine oxidase (MAO) activity can regulate levels of biogenic amines and neuronal activity in the nervous system. The two types of MAO activity, A and B, appear to have different domains of activity in the body. Brain tissue has both types of activity, although adrenergic neurons are thought to contain exclusively MAO-A. MAO activity can also be measured in peripheral tissues: MAO-A in cultured skin fibroblasts and placenta, and MAO-B in platelets and lymphocytes. These two types of activity are mediated by different enzyme molecules and are regulated independently by endogenous and exogenous factors including genetic determinants, hormones, and aging. In humans, inhibition of MAO-A activity leads to mood elevation in depressed patients; in contrast, low MAO-B activity in platelets has been associated with an increased susceptibility to psychopathology. In order to assess further the role of MAO activity in human mood and behavior, it will be important to measure both forms of the enzyme independently and to establish correlations between levels of activity and discrete phenotypic traits.

Characterization of monoamine oxidase activity during early stages of quail embryogenesis.

Catecholamines and other biogenic amines may play a role in early embryogenesis in addition to functioning as neurotransmitters after neuronal differentiation. Regulation of amine levels is mediated by several different parameters including activity levels of degradative enzymes. Since monoamine oxidase (EC 1.4.3.4) is the primary degradative enzyme for these biogenic amines, we have begun to characterize MAO activity during quail embryogenesis. Our results demonstrate that MAO activity is present at all stages of development examined (stages 2–22) and that the MAO specific activity levels are highest during the earliest stages (stages 2–6). Two types of MAO activity similar to adult avian and mammalian MAO‐A and MAO‐B have been demonstrated by differential clorgyline sensitivity of tryptamine deamination. In addition, SDS‐PAGE of embryonic quail [3H]pargyiine‐labeled MAO demonstrates that the quail MAO‐A and MAO‐B flavin‐containing subunits have apparent molecular weights of 63,000 and 62,000 respectively.

Expression of IGF-II, the IGF-II/Mannose-6-Phosphate Receptor and IGFBP-2 During Rat Embryogenesis

It is becoming increasingly apparent that the IGF system (including both IGF-I and IGF-II, at least two IGF receptors, and a family of at least six IGF binding proteins; 1-3) has a fundamental role in the normal progression of prenatal development. The initial observation that IGF-II peptide levels were high in the fetus but decreased post-natally (4) was the first to suggest that this peptide might have functional importance during ontogeny. In situ hybridization studies (5-7) extended Northern analysis and confirmed that the developmental regulation of IGF-II expression extends to the level of RNA regulation and demonstrated that the IGF-II gene expression pattern during ontogeny is precisely regulated both temporally and spatially. These results suggested that if the autocrine-paracrine mode of action classically proposed for the IGFs (1,2) was in fact correct, then interference with normal IGF-II synthesis would be expected to produce regional rather than systemic deficits.

Molecular and Immunocytochemical Studies of Neurofibromas and Related Cell Types

Abnormal proliferation of cell types normally associated with peripheral neurons is a central feature of neurofibromatosis. Although the molecular basis underlying the localized development of dermal neurofibromas is not known, it is conceivable that abnormal expression of growth factor genes or oncogenes directs this proliferation At present nerve growth factor (NGF), which has well-defined effects on specific populations of embryonic and adult cells (see References 1-4 for reviews), has been the most extensively studied growth factor. Despite extensive biochemical characterization of this molecule and molecular studies of its effects on NGF-responsive cells, relatively little is known about its relevance to specific disease states. Although linkage analysis has strongly suggested that a structurally altered P-NGF gene is not correlated with neurofibromatosis’ (but see Reference 6), the possibility remains that altered regulation or processing of P-NGF in neurofibroma tissue may be related to the development of this condition. Recent, but indirect, evidence has offered some support for this possibility. Some sympathetic nerve endings exhibit hypertrophy and fail to innervate appropriate target organs following development of dermal neurofibromas.7 Although it is not known

Differences in monoamine oxidase activity between cultured noradrenergic and cholinergic sympathetic neurons

Two types of monoamine oxidase activity (MAO-A and MAO-B) help regulate the levels of biogenic amines such as catecholamines and serotonin. Although MAO-A has greater activity toward most catecholamines than MAO-B, no direct experiments have determined the types and levels of MAO activity that are normally expressed in noradrenergic neurons. Noradrenergic neurons from neonatal rat superior cervical ganglia were isolated and cultured under conditions that permit either continued expression of the noradrenergic phenotype or promote a transition to a predominantly cholinergic phenotype. After 14-21 days in vitro, neurons from both types of cultures were assayed for the type and amount of monoamine oxidase activity using tryptamine, a common substrate for both MAO-A and MAO-B. Neurons cultured under noradrenergic conditions expressed sevenfold greater MAO activity than neurons cultured under cholinergic conditions. Essentially all MAO activity in the noradrenergic cultures was inhibited by preincubation with 10(-8)-10(-9) M clorgyline, which indicated that this activity was primarily MAO-A. Cultures grown under cholinergic conditions exhibited 6- to 10-fold lower MAO-A activity and an 8- to 10-fold lower level of catecholamine synthesis from labeled precursors compared to neurons grown under noradrenergic conditions. These results directly demonstrate that high MAO-A activity is expressed in noradrenergic neurons in vitro. The corresponding decreases in both MAO-A specific activity and catecholamine synthesis as neurons become cholinergic in vitro suggest that the expression of the noradrenergic phenotype involves the coordinate regulation of degradative as well as synthetic enzymes involved in catecholamine metabolism.

Biochemical and genetic analysis of MAO A and B

Monoamine oxidase (MAO) (monoamine: O2 oxidoreductase EC 1.4.3.4.) is located in the mitochondrial outer membrane and is the enzyme primarily responsible for the degradation of catecholamines and other biogenic amines in the nervous system (Houslay et al., 1976; Murphy, 1978). The enzyme is believed to be comprised of two subunits of approximate molecular weight 60,000, with one subunit containing a covalently bound flavin co-factor (Minamiura and Yasunobu, 1978; Salach, 1979). Two types of MAO activity, A and B, have been defined by characteristic substrate preferences and inhibitor sensitivities (Johnston, 1968; Garrick and Murphy, 1980).