Hydrolysis of substance P in the rabbit retina: I. Involvement of acetylcholine and acetylcholinesterase. An in vivo study

Abstract

The laminar patterns of acetylcholinesterase (AChE) activity and substance P (SP) immunoreactivity within the inner plexiform layer (IPL) of the rabbit retina show striking similarities. Discrete bands of SP-immunoreactivity were seen at 1–7%, 40–48% and 85–95% depth of IPL. AChE activity was present throughout the entire thickness of the IPL with moderately stained bands in each sublamina (3–24% in sublamina a and 62–89% in sublamina b depth IPL). These bands were bordered on both sides by bands of even greater density (in sublamina a 0–3% and 24–34% and in sublamina b 55–62% and 89–100% depth IPL). Cell processes staining for choline acetyltransferase (ChAT) have previously been shown to ramify at 19–24% and 63–79% depth levels (38). Thus, SP- and ChAT-immunoreactive bands are located in both sublaminae, positioned within regions of moderate AChE activity and flanked by bands with greater AChE activity. This strong morphological correspondence and reported interactions between acetylcholine (ACh), AChE and SP (23) in vitro provide the basis for the present study to determine whether such interactions can be demonstrated in vivo. Retinas infused with ACh showed a 60% average increase in SP-IR as compared with untreated retinas from the same animals. Treatment with diisopropylfluorophosphate (DFP) also resulted in a 56% increase in SP-IR. The ability of ACh to induce increased levels of SP was not inhibited by CoCl 2, atropine or mecamulamine, ruling out the possibilities of polysynaptic transmission or involvement of muscarinic or nicotinic receptors. Infusion of ACh did not increase the levels of preprotachykinin-mRNA indicating that the increase in SP-IR is not due to de novo synthesis but rather to inhibition of the enzyme(s) responsible for SP degradation. Whether AChE functions alone or in concert with other enzymes to hydrolyze SP cannot be determined from these experiments but is addressed in a separate study (48).