Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands.
The rabbit has been a useful model for in vivo studies of the pharmacologic control of lacrimal gland fluid secretion. However, by contrast with rodent exorbital lacrimal glands, the rabbit lacrimal gland has not been subjected to detailed cellular, subcellular, or biochemical analyses. Procedures were developed to isolate rabbit lacrimal acini by collagenase digestion and mechanical dispersion. The preparations exhibited good morphology, and trypan blue exclusion rates generally exceeded 90%. The isolated acini responded to carbachol by releasing protein and increasing Na+ unidirectional influx rates. The presence of muscarinic cholinergic and beta-adrenergic receptors was indicated by specific binding of the muscarinic cholinergic antagonist, 3H-N-methylscopolamine (3H-NMS; dissociation constant, Kd, 0.55 nmol/l), and the beta-adrenergic antagonist, 3H-CGP12177 (Kd, 0.34 nmol/l). The maximal binding values measured in crude membrane preparations were 79 fmol/mg for 3H-NMS and 40 fmol/mg for 3H-CGP12177. Subcellular fractionation analyses showed various membrane populations, including a series of Golgi-derived populations admixed with a major endoplasmic reticulum-derived population, a population that may represent the basal-lateral plasma membranes, and a series of populations with characteristics suggesting they are involved in the assembly or recycling of basal-lateral membrane constituents. The authors believe the ability to isolate and analyze acinar preparations from the rabbit lacrimal gland will facilitate various studies of acinar cell biochemistry and physiology that would be impractical with the relatively smaller amounts of material that can be obtained from rat or mouse exorbital lacrimal glands.
Bradley
M E
ME
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Lambert
R W
RW
Lambert
R W
RW
Lee
L M
LM
Mircheff
A K
AK
D000818:Animals; D001665:Binding Sites; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007765:Lacrimal Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D011817:Rabbits; D011869:Radioligand Assay; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013347:Subcellular Fractions
D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase