Ultracytochemical demonstration of ATP-dependent calcium pump in ameloblasts of rat incisor enamel organ.

Abstract

The enamel organ of the growing rat incisor was fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of Ca- and Mg-activated membrane ATPase by a one-step lead technique at alkaline pH. To inhibit nonspecific alkaline phosphatase, 5 mM levamisole was added to the incubation media. Intense Ca- and Mg-ATPase activity was demonstrated in the cell surfaces of the secretory ameloblasts, except at the proximal and distal junctional complexes and the gap junctions in the lateral and basal cell surfaces. Deep plasma membrane invaginations at the proximal and distal parts of Tomes processes facing interrod- and rod-enamel growth regions exhibited the strongest enzymatic reaction. Mg-ATPase activity was also shown to be present in the plasma membranes of secretory ameloblasts but it was less intense than Ca-ATPase. Except for a slight reaction in the Golgi membranes, all other cell organelles of the secretory ameloblasts and the adjacent enamel matrix were free of enzymatic reaction. However, when the tissues were incubated in media lacking levamisole, a prominent enzymatic reaction was observed in the newly secreted enamel matrix of the rod and interrod growth regions as well as on the plasma membranes of the cells. In maturation ameloblasts of both ruffle-ended and smooth-ended types, a weak reaction for Ca- and Mg-ATPase was restricted to basal cell surfaces facing the papillary cell layer. In tissues incubated in media lacking levamisole, a variable deposition of reaction products was observed in the Golgi membranes, mitochondrial membranes, tubular elements of smooth endoplasmic reticulum in the ruffled border zone, and along the plasma membranes of the ruffled border. Throughout the secretory and maturation stages, a moderate and/or weak enzymatic reaction for both Ca- and Mg-ATPase was seen in the plasma membranes of the cells of the stratum intermedium and the papillary layer when incubated in media with levamisole. Omission of substrate ATP and/or the enzyme activator CaCl2 from the incubation media for Ca-ATPase produced a negative reaction in the tissues examined. When the calmodulin blocker trifluoperazine was administered to the rats intravenously, Ca-ATPase activity was almost completely abolished from the plasma membranes of secretory ameloblasts, but not of other cell types.