Winifred A. Koelle

HISTOCHEMICAL EVIDENCE AND CONSEQUENCES OF THE OCCURRENCE OF ISOENZYMES OF ACETYLCHOLINESTERASE

Frozen sections of unfixed cat stellate (StG) and ciliary (CG) ganglia and intercostal muscle (ICM) and mouse ICM were exposed to saline (0.9% NaCl) solution and to 0.3% Triton X-100 in saline solution, and then stained for acetylcholinesterase (AChE) by a modification of the standard copper-thiocholine method. In the StG and CG, treatment with saline solution resulted in marked diffusion of AChE, and Triton X-100 caused its marked loss from the CG and nearly complete loss from the StG. In ICM, in contrast, both treatments caused increased intensity of staining of AChE at the motor end plates (MEPs). All of the foregoing effects were reversed progressively, with concomitant reduction in AChE activity, by prior fixation in cold formaldehyde (4%)-sucrose (7.5%)-maleate buffer (pH 7.3) solution for 2-24 hr. Results are interpreted on the basis that the AChE of the ganglia and MEPs exists predominantly in different isoenzymatic forms, the possible natures of which are discussed.

Effects of inactivation of butyrylcholinesterase on steady state and regenerating levels of ganglionic acetylcholinesterase.

The effects of single and repeated injections of tetramonoisopropyl pyrophosphortetramide (iso‐OMPA), a selective inactivator of butyrylcholinesterase (BuChE), were studied on the ganglionic and muscular levels of BuChE and acetylcholinesterase (AChE) in cats during the steady state and following the irreversible inactivation of both enzymes by isopropylmethylphosphonofluoridate (sarin). Single intravenous injections of iso‐OMPA, 3.0 or 6.0 μmol/kg, produced nearly total inactivation of BuChE with no immediate effect on the AChE of the superior cervical (SCG), stellate (StG), and ciliary (CG) ganglia and inferior oblique (10) muscle; regeneration of BuChE occurred at approximately the same rate in the three ganglia, and at 4–6 days the AChE levels were significantly elevated. When single doses of iso‐OMPA were given 1 h following sarin, 2.0 μmol/kg, intravenously, there was a slight increase in the rate of AChE regeneration during the ensuing 2 days. With the repeated injection of iso‐OMPA, 3.0 μmol/kg every 48 h, there was a consistent but not statistically significant reduction in AChE regeneration at 4, 6, 12, and 18 days following sarin in all 3 ganglia. Similar treatment with iso‐OMPA alone produced significant increases in ganglionic AChE at all these periods excepting the longest. The daily injection of iso‐OMPA for 6 days, which maintained ganglionic BuChE at approx 2% of the control values, produced significant reductions in AChE regeneration, but again significant increases in ganglionic AChE levels in cats that did not receive sarin. The IO muscle did not exhibit these effects. A working hypothesis is proposed, that BuChE is a precursor of ganglionic AChE, and that the level of BuChE participates in the regulation of AChE synthesis by inhibition of a preceding rate‐limiting step.

Selective, near-total, irreversible inactivation of peripheral pseudocholin-esterase and acetylcholinesterase in cats In vivo

Abstract Tetramonoisopropyl pyrophosphortetramide (Iso-OMPA) was confirmed to be a highly selective inactivator of cat pseudocholinesterase (BuChE) over wide ranges of concentration and temperature in vitro ; intravenous doses of 3.0 or 6.0 μmoles (approximately 1.0 or 2.0 mg)/kg produced nearly total inactivation of peripheral BuChE, with no detectable inactivation of acetylcholinesterase (AChE). Selective inactivation of AChE in vivo was obtained as follows: after a dose of mephentermine, 2.0 to 3.0 mg/kg, i.v., to sustain blood pressure, cats received an intravenous dose of 10-(α-diethylaminopropionyl) phenothiazine HCl (Astra 1397) up to 200 μmoles (73 mg)/kg, which produces selective reversible inhibition of BuChE; they were then given 1.0 μmole (0.332 mg) 2-diethyoxyphosphinylthioethyldimethylamine acid oxalate (217 AO)/kg, which is sufficient when given alone to produce essentially total, irreversible inactivation of AChE and BuChE. Under these conditions, approximately one-third of the autonomic ganglionic BuChE, but none of the AChE, was protected and restored to activity. Quantitative results, obtained by a spectrophotometric method, were confirmed histochemically.

EFFECTS OF PROTECTION OF BUTYRYLCHOLINESTERASE ON REGENERATION OF GANGLIONIC ACETYLCHOLINESTERASE

The regeneration of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was followed in the superior cervical (SCG), stellate (StG), and ciliary (CG) ganglia and inferior oblique (IO) muscle of cats for the 3‐day period following their inactivation by isopropylmethylphosphonofluoridate (sarin; 2.0 μmol/kg intravenously), with and without preservation of over half the butyrylcholinesterase activity by the prior intravenous infusion of 10‐(α‐diethylaminopropionyl) phenothiazine HCl (Astra 1397; 100 or 200 μmol/kg). Rates of regeneration of AChE in the three ganglia exhibited the same sequence as their relative proportions of AChE‐containing cholinergic ganglion cells (SCG < StG < CG); no such difference was found in the rates of butyrylcholinesterase regeneration. The mean AChE levels in all three ganglia were higher at 48 h in the cats that had received Astra 1397 (100 μmol/kg) prior to sarin. This finding is interpreted as evidence that BuChE may function as a precursor in the synthesis of AChE.

EFFECTS OF ALDEHYDE FIXATION AND OF PREGANGLIONIC DENERVATION ON ACETYLCHOLINESTERASE AND BUTYROCHOLINESTERASE OF CAT AUTONOMIC GANGLIA

Fixation of cat stellate ganglia by perfusion in situ with cold 4% formaldehyde in Krebs-Ringer solution, followed by immersion in the same solution for 3 or 6 hr, produced inactivation of approximately one-half the acetylcholinesterase (AChE) and butyrocholinesterase (BuChE); after 21 hr, further inactivation occurred. Similar fixation with glutaraldehyde (1, 2 and 4%) inactivated over three-quarters of the AChE and BuChE at 3 hr, but there was little further loss after 21 hr. Following sectioning of the preganglionic trunk, the AChE activity of the cat superior cervical ganglion fell to approximately 20%, and BuChE fell to approximately 50%; this confirms an early report by Sawyer and Hollinshead, which has been questioned in part because of subsequent histochemical findings. The basis for the discrepancy is discussed, along with its bearing on the interpretation of histochemical observations in general.

Effects of Selective Alkylphosphorylation of Propionylcholinesterase on the Regeneration of Acetylcholinesterase in the Aqueous Soluble Fraction of Superior Cervical Ganglia of the Rat Following Sarin

Abstract: The rates of regeneration of acetylcholinesterase (AChE) and propionylcholinesterase (PrChE) in the supernatants of aqueous homogenates of rat superior cervical ganglia, centrifuged at 100,000 g for 90 min, were determined at 1,3, 6, and 16 h following their inactivation (>90%) by administration of sarin, 2.0μmol/kg i.v. Values were compared with those in animals in which the PrChE was continually suppressed by the repeated, fractional administration of iso‐OMPA, in a total dose of 10 or 20 μmol/kg i.p. These doses of iso‐OMPA alone produced 96–99% inactivation of PrChE with no detectable effect on AChE. Significant suppression of AChE regeneration by iso‐OMPA administration was noted only at 6 h; in contrast with earlier findings in the cat, administration of iso‐OMPA alone caused no significant increase in ganglionic AChE activity.

Histochemical and Pharmacological Evidence of the Function of Butyrylcholinesterase

Nearly 40 years ago Alles and Hawes (1) demonstrated that the cholines-terases (ChE) of erythrocytes and plasma differ. The work of Mendel and associates (26, 27), Nachmansohn (29), Augustinsson (2, 3) and others clearly established the distinguishing characteristics between acetylcholinesterase (also called specific, or true ChE, ACh hydrolase; EC3.1.1.7; A ChE) and butyrylcholinesterase (non-specific or pseudo-ChE, acylcholine acyl-hydrolase; EC 3.1.1. 8; BuChE). The former enzyme predominates in the central nervous system (CNS), the motor endplates (MEPs) of skeletal muscle, and erythrocytes, and the latter in many types of smooth muscle, the liver and plasma; both enzymes occur in high concentrations in autonomic ganglia (16). The substrate concentration-velocity of hydrolysis curves for ACh and other Ch esters with A ChE are bellshaped, with the peaks at approximately 0. 003 M. ACh is hydrolyzed most rapidly, methacholine (MeCh) at about one-third and butyrylcholine (BuCh)at only a small percent of the rate of ACh, and benzoylcholine (BzCh) insignificantly. BuChE shows typical sigmoid, Michaelis-Menten type substrate-velocity curves, with the highest rate for BuCh, followed by ACh and BzCh, and practically no hydrolysis of MeCh.

Questions Raised by the Electron Microscopic Localization of Acetylcholinesterase and Butyrylcholinesterase in Normal and Denervated Superior Cervical Ganglia in the Cat

With the development of the copper thiocholine (CuThCh) histochemical method for the light microscopic (LM) localization of cholinesterases over 30 years ago (15) and the application of its modifications (9–11,16) to the normal and preganglionically denervated superior cervical ganglion (SCG) of the cat, the distributions of acetylcholinesterase (AChE) and butyrylcholinesterase (Bu-ChE) in this tissue appeared to have been established. In the normal ganglion, AChE was present in high concentrations in the perikarya of occasional (< 1%) ganglion cells (6) and in traces in the remainder, and in considerable concentration throughout the neuropil (Fig. 1). Following preganglionic denervation, staining for AChE disappeared completely from the neuropil but remained unchanged in the perikaya of the ganglion cells. It was therefore concluded that staining of the neuropil represented enzyme confined solely to the preganglionic fibers and their terminals. Staining for BuChE was intense throughout the neuropil and completely absent from the perikaya of the ganglion cells; following denervation, there was only a slight decrease in the intensity of staining. From these and related findings (1,2) it appeared that BuChE was present only in capsular glial cells. These findings were consistent with the results of quantitative determinations in normal and preganglionically denervated ganglia (14,25). Pharmacological studies provided a hypothetical explanation for the function of the presynaptically located AChE (12,30).