Anne Tormay

Detection of Anti-Acetylcholine Receptor Factors in Serum and Thymus from Patients with Myasthenia Gravis

Since the blood and thymus of patients with myasthenia gravis may contain inhibitors of neuromuscular transmission that affect acetylcholine receptors of striated muscle, we used denervated rat muscle to test for inhibitors in 43 serums and 18 thymus glands from such patients. Seven per cent of serums inhibited the binding of 125l alpha-bungarotoxin to triton-solubilized receptors; 65 per cent interfered with binding of toxin-labeled receptors to concanavalin-A coupled to Sepharose gel, and 85 per cent formed IgG-receptor complexes detectable by immunoprecipitation. Serum inhibitory activity varied widely among patients with similar clinical manifestations and was not correlated with duration of myasthenia gravis or thymectomy. Among thymus extracts, 44 per cent were inhibitory in the concanavalin-A binding assay, whereas 72 per cent contained anti-receptor IgG. Thus, serums from patients with myasthenia gravis contain more than one anti-receptor factor.

Alpha-adrenergic antagonists: Correlation of the effect on intraocular pressure and on α2-adrenergic receptor binding specificity in the rabbit eye

Six alpha-adrenergic antagonists, which have a range of selectivities for alpha 1- and alpha 2-adrenoreceptor subtypes, were compared with respect to their ability to reduce intraocular pressure (IOP) after topical application to the rabbit eye, and their affinity and selectivity for alpha 2-adrenoreceptors, as determined by binding to membranes prepared from rabbit iris-ciliary body. A routine assay for alpha 2-adrenoreceptors using [3H]-rauwolscine was developed for this purpose. ICB contained 200-300 fmol (mg protein)-1 alpha 2-adrenoreceptors which represents approximately two-thirds of the total number of alpha-adrenoreceptor sites present in this tissue. All six antagonists bound at alpha 2-adrenergic receptor sites in an apparently simple competitive manner. The Kd for three of the drugs was about 10 nM (rauwolscine, yohimbine, WB-4101) and the Kd for the other three was greater than 3500 nM (prazosin, corynanthine, thymoxamine). However, all six antagonists were effective ocular hypotensive agents when given topically in a 50 microliter dose of 1% (w/v) concentration. The ability of alpha-adrenergic antagonists to lower IOP in the rabbit did not correlate with a single alpha-receptor subtype and appears to involve at least two separate mechanisms, one mediated by alpha 2-adrenergic receptors and one mediated by alpha 1-adrenergic receptors.

Desensitization of the β-adrenergic receptor-adenylate cyclase complex in rabbit iris-ciliary body induced by topical epinephrine

Topical treatment of the rabbit eye with three successive doses of 2% epinephrine bitartrate alters the in vitro drug response of the β-adrenergic receptor-adenylate cyclase enzyme complex in membrane fragments prepared from the iris and ciliary body. Treated tissues showed a significant decrease in activation of adenylate cyclase by guanosine triphosphate (100 μ m ), guanosine 5′-diphospho-imido phosphate (0·2 μ m ), and by isoproterenol (10 μ m ) in presence of either of the above guanyl nucleotides but showed no significant change in basal adenylate cyclase activity or in activity stimulated by fluoride (10 m m ). The epinephrine-induced desensitization of β-adrenergic receptor-coupled adenylate cyclase is associated with a functional impairment of the guanyl nucleotide regulatory protein and possibly with a small decrease in the number of affinity of β -adrenergic receptor ligand binding sites. Agonist-induced changes in receptors and in their molecular effector systems represent basic mechanisms for alteration of tissue responses to drugs and may thus be involved in the therapy of glaucoma with chronic topical epinephrine.

Role of G-proteins in ciliary process adenylyl cyclase responses of the albino rabbit eye

After intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5 -1 microgram/eye) into the albino rabbit eye, the in vitro responses of ciliary process adenylyl cyclase (AC) to isoproterenol, vasoactive intestinal peptide (VIP), and forskolin (FSK) were increased 21-40% for PTX, but for CTX-injected eyes AC responses to fluoroaluminate, VIP and FSK decreased 70-50%. The increased responses after PTX suggests that this toxin blocked an inhibitory Gi control of AC that is present in the control tissue. However, prolonged (> 24 hr) in vivo exposure to CTX appears to down-regulate the AC enzyme. In contrast to the in vivo findings, AC responsiveness was unaffected by PTX pre-treatment of membranes in vitro, while CTX pre-treatment increased basal activity (+600%), and the FSK response (+30%), but decreased responsiveness to fluoroaluminate, VIP and isoproterenol by 88-56%. Treatment of ciliary process membranes with 32P-NAD and CTX or PTX followed by SDS-PAGE autoradiography of labelled proteins gave two bands for the G-protein alpha-subunits of Gs (45, 56 kDa) and one broad band centered at 40 kDa for Gi-type subunits respectively. Western blots using specific antibodies showed the presence of Gi type I or III, but no detectable Gi type II or Go in rabbit ciliary processes. We conclude that the changes in adenylyl cyclase enzyme responses after intraocular CTX or PTX may not correlate with cAMP levels and intraocular pressure effects. However, the in vitro biochemical data on AC responses and on G-proteins provide evidence for dual regulation of ciliary process AC by activating and inhibitory G-proteins.

Endotoxins in cholera and pertussis toxins interfere with in vivo responses to these agents in the albino rabbit eye.

Intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5-1 microgram/eye) decreased intraocular pressure (IOP) up to 50% in the albino rabbit eye, which lasted up to six days. Both toxins were active on G-proteins as determined by in vitro and in vivo effects on ciliary process adenylyl cyclase activity and by ADP ribosylation of G-protein alpha-subunits with 32P-NAD. However, forty-two hours after toxin injection aqueous humor proteins increased from control levels of 0.8-1.2 mg/ml to 8-25 mg/ml. Both toxins contained 1-3 parts per thousand endotoxin sufficient to cause the IOP and aqueous humor protein responses observed. We conclude that the in vivo responses to intraocular CTX or PTX obtained from commercial sources may not provide unequivocal evidence for the role(s) of G-proteins in aqueous humor dynamics, and must be interpreted with caution.