Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Distances between functional sites in cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase. Inter-lanthanide energy transfer

The high-affinity Ca2+-binding sites of cardiac sarcoplasmic reticulum (Ca2+ +Mg2+)-ATPase have been probed using trivalent lanthanide ions. Non-radiative energy-transfer studies, using luminescent probe Eu3+ as a donor and Nd3+ or Pr3+ as acceptor, were carried out to estimate the distance between two high-affinity Ca2+-binding/transport sites. Eu3+ was excited directly with pulsed laser light and the energy-transfer efficiency to Nd3+ or Pr3+ was measured, under the conditions in which most donor-acceptor pairs occupied the high-affinity Ca2+ sites. The distance between two high-affinity Ca2+ sites is about 0.89 nm. In the presence of ATP the distance between the high-affinity sites is about 0.855 nm, whereas in the presence of adenosine 5'-[beta, gamma-methylene]triphosphate or adenosine 5'-[beta, gamma-imino]triphosphate the distance is about 0.895 nm. To estimate the distance between the high-affinity Ca2+ sites and ATP-binding/hydrolytic site, we have measured the energy-transfer efficiency between Eu3+ and Cr3+-ATP with Eu3+ at the high-affinity Ca2+ sites and Cr3+-ATP at the ATP-binding/hydrolytic site. Our results show that ATP-binding/hydrolytic site is separated by about 2.2 nm from each high-affinity Ca2+ site.     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Vit C.Fe(III) induced loss of the covalently bound phosphate and enzyme activity of phosphoglucomutase

Rabbit muscle phosphoglucomutase was irreversibly inactivated upon preincubation with vitamin C (Vit C). Fe(III), NADH.NADH oxidase.Fe(III), or ferritin.Vit C. Substrate, glucose 1-phosphate and Mg2+ afforded partial protection. No altered amino acid could be detected in the inactive enzyme. Enzyme so inactivated was more susceptible to trypsin. More importantly, during inactivation, the enzyme lost up to 70% of its enzyme-bound phosphate; the completely inactivated enzyme retained the remainder of the bound phosphate which was isolatable as phosphoserine residing in the 22-amino acid long tryptic peptide. Free phosphoserine as well as those in phosphorylase alpha and phosphocasein were resistant to the oxidizing system, suggesting that the phosphoserine of phosphoglucomutase is uniquely vulnerable to these treatments. Alternatively, a fraction of the total 1 mol of phosphate in the phosphoform of phosphoglucomutase may not be associated with phosphoserine. Phosphoglyceromutase, which has phosphohistidine at its active site, was also inactivated by the oxidizing system. However, it did not release any of the bound phosphate.     

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

Monoclonal antibodies to mitochondrial coupling factor B

Two monoclonal antibodies (MAb I and IV) have been prepared which showed high and specific reactions towards bovine heart mitochondrial coupling factor B (FB). Both have been identified as sub-type IgG1 of mouse immunoglobulins. MAb I reacts with purified and functionally active FB, alkylated or oxidized forms of FB and even with peptides formed on digestion of FB with trypsin. When used together, MAb I and IV reacted with FB in immunoblots of normal and urea treated samples of mitochondria, submitochondrial particles, ammonia-EDTA extracted particles, and H+-ATPase. Both MAbs inhibited FB-stimulated ATP-dependent reverse electron flow activity when FB was incubated with the antibody either before or after its addition to FB-deficient AE-particles. Reactivity of MAb I towards FB declined upon exposure of FB to guanidine HC1 while reactivity of MAb IV remained unaltered.     

On control of the function of the sodium-potassium pump in malignant cells

This paper introduces a double-layer enzyme-membrane model representing the Na⁺−K⁺ pump in living cells. We present a mathematical solution to the problem of controlling the sodium flux in malignant cells, where an inhibitor exists in the outer layer of the membrane. We give an algorithm for the numerical resolution of this problem of optimal control with illustrations. Finally, we point out the biological importance of this study.