Mouse megakaryocytes secrete acetylcholinesterase

Abstract

Acetylcholinesterase (AchE), an essentially specific and early marker of rodent thrombocytic series, in several tissues acts both as a constituent of the cellular membrane and as a secretory enzyme. This study presents the ultrastructural transport and fate of this substance in the megakaryocytes of mouse bone marrow, using Tranum-Jensen and Behnke's adaptation of the indirect thiocholine method. It is shown that megakaryoblasts and megakaryocytes undergo a typical secretory cycle consisting of (1) enzyme synthesis and segregation on the endoplasmic reticulum and nuclear envelope, (2) enzyme concentration in AchE-vesicles and AchE-granules formed from the cisternae of the Golgi apparatus, and (3) discharge in the demarcation membrane system and extracellular space. The AchE-vesicles and granules appear to be hitherto unrecognized megakaryocytic organelles as they do not resemble alpha nor the dense granules, and their mode of formation and fate differ from those of primary lysosomes and peroxidase granules. Released platelets reveal AchE activity in the open canalicular system. The data are compatible with the hypothesis that by controlling acetylcholine concentration in hematopoietic tissues, the secretion of AchE by megakaryocytes can modulate the proliferative activity of megakaryocytes progenitors.

Flow cytometric analysis of megakaryocyte differentiation

“…In studies reported elsewhere (221, we have sorted these different megakaryocyte subpopulations for analysis of their metabolic capacity to synthesize thromboxane B2, which we have suggested previously (21) may serve as a potential metabolic marker of differentiation. In future studies we plan to investigate the complexities raised by the results of the present study; to do so, we will use monoclonal antibodies directed to megakaryocyte subpopulations and platelets as well as make fluorescent measurements of acetylcholinesterase activity (10,15), parameters that may prove useful as additional probes of megakaryocyte differentiation.…”

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase

“…To inhibit cholinesterase activity, 0.2 mM DFP (final concentration) a serine protease inhibitor, or 0.2 mM (final concentration) 1,5-bis (4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, a specific AchE inhibitor (BW248C51; Sigma; Paulus et al, 1981) was diluted in the Tris-Triton-DTNB solution and added to each well.…”

“…Since previous workers have reported that a small quantity of AchE is present in murine red cells (Paulus et al, 1981), platelet-free red cells were prepared as described and assayed for AchE activity. No significant activity was noted using concentrations of red cells whose intrinsic OD due to hemoglobin was higher than is ever observed in marrow cultures (data not shown).…”

The regulation of megakaryocyte and platelet production

“…This finding has led to the hypothesis that autoregulation of megakaryocytes in rodents is controlled by acetylcholinesterase. Data presented by Puulus et al [26] support this hypothesis, i.e., the secretion of acetylcholinesterase by megakaryocytes was thought to control the proliferation of megakaryocyte progenitors. The injection of Neostigmine (an inhibitor of acetylcholinesterase) into mice elevated the percentage of small acetylcholinesterase-positive (SAChE+) cells [25].…”

“…Megakaryocytopoiesis is clearly regulated, to a large degree, by the number and/or function of circulating platelets. However, evidence also exists for autoregulation of megakaryocytopoiesis [l, [22][23][24][25][26]. It has been observed that increased numbers of mature megakaryocytes in the marrow of rodents will decrease the number of megakaryocytic progenitors, and megakaryoqtic macrocytosis occurs in several conditions associated with low numbers of megakaryocytes [23].…”