The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in stentor coeruleus

Abstract

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N6, Z'-O-dibutyryl adenosine cyclic 3':5'-monophosphate (DBC) or theophylline begun a t the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3':5'-cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions.To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.

Phytohemagglutinin Induces Delays in Oral Regeneration in the Ciliate Stentor coeruleus1

“…First, if there were toxic side effects, they were probably not due to the fact that high drug concentrations were used. Protozoans, particularly Stentor, seem to be less sensitive to drug concentrations effective on metazoan cells (25). The concentrations of PHA used in this study are very similar to lectin concentrations used with other eukaryotic microorganisms, such as Tetrahymena pyriformis (1 3, 3 1, 35), Blepharisma intermedium (30), Stylonychia mytiius (1 2), and Dictyostelium discoideum (6).…”

Concanavalin a inhibits oral regeneration in Stentor coeruleus through a mechanism involving binding to the cell surface

“…In almost all cases the presence of excess extracellular calcium can negate the effects of a particular agent on oral regeneration, suggesting the necessity of extracellular calcium for normal oral regeneration. However, the fact that the removal of extracellular calcium alone does not affect oral regeneration complicates this conclusion (Maloney and Burchill, 1977). Still, the implication of the results with Con A and PHA, as well as some other membrane active agents, is that calcium ions have some connection with the action of such agents.…”

“…Culture methods Stentor were cultured as described previously (Maloney and Burchill, 1977). Basically cells were grown in 2-3-quart Pyrex baking dishes containing a synthetic salt solution composed of 0.55 mM CaC12, 0.15 mM MgS04, 0.75 mM NaC03, and 0.33 mM KHCO3, with the pH adjusted to 7.5 with HC1.…”

Identification and Localization of a Protein Immunologically Related to Caltractin (Centrin) in the Myonemes and Membranelles of the Heterotrich Ciliate Stentor coeruleus

“…Stentor coeruleus was originally obtained from a pond on the Butler University campus and has been cultured in the laboratory since that time as described previously (Maloney and Burchill 1977). Briefly, cells were cultured in Pyrex baking dishes in a synthetic salt solution containing 0.55 mM CaCl 2 , 0.15 mM MgSO 4 , 0.75 mM Na 2 CO 3 , and 0.33 mM KHCO 3 made up in deionized water, with the pH adjusted to 7.5 with HCl.…”