Cyclic GMP levels and guanylate cyclase activity in pheromone-sensitive antennae of the silkmoths antheraea polyphemus and Bombyx mori

Abstract

Female sex pheromones applied to freshly isolated, living antennae of male Antheraea polyphemus and Bombyx mori led to an increase of cGMP. A 1:1 mixture of 2 pheromone components of Antheraea polyphemus blown for 10 sec in physiological concentrations over their antennal branches raised cGMP levels about 1.34-fold (±0.08 SEM, n = 23) from a basal level of 3.0 ± 0.6 (SEM, n = 20) pmol/mg protein. Similarly, bombykol elicited a 1.29-fold (±0.13 SEM, n = 23) cGMP increase in antennae of male Bombyx mori from a basal level of 2.7 ± 0.5 (SEM, n = 24) pmol/mg protein. No cross-sensitivity was found with respect to pheromones from either species. In antennae of female silkmoths, the cGMP response was missing upon stimulation with their own respective pheromones according to the known lack of pheromone receptor cells in the female. cAMP levels in the male antennae of 14.2 ± 2.9 (SEM, n = 4) pmol/mg protein in A. polyphemus and 15.0 ± 3.0 (SEM, n = 5) pmol/mg protein in B. mori were not affected by pheromone stimulation. Within 1-60 sec, the extent of cGMP increase in B. mori was independent of the duration of pheromone exposure. The levels of cGMP in pheromone-stimulated antennae of both species remained elevated for at least 10 min, i.e., much longer than the duration of the receptor potential measured in single-cell recordings. Guanylate cyclase activity was identified in homogenates of male and female antennae from both species. The Km of the guanylate cyclase from male B. mori for the preferential substrate MnGTP was 175 μM. Specific activity at the pH optimum of 8.25 was 5-30 pmol × min-1 × mg-1. About half of total guanylate cyclase activity was soluble. SH-group blocking agents inhibited, whereas dithiothreitol and glutathion doubled enzyme activity. The enzyme was inhibited by antibodies raised against soluble guanylate cyclase from bovine lung. To localize the increase of cGMP in the antennae, sensory hairs of male A. polyphemus which contain only the receptive dendrites of the olfactory neurons, were isolated. Neither changes in cGMP concentrations upon pheromone stimulation nor guanylate cyclase activity were found in the den-drite preparation. This indicates that cGMP is possibly produced within the soma of the olfactory neurons or within the surrounding auxiliary cells. This study leads to the conclusion that cGMP is not involved in the transduction process generating the receptor potential. Both the down-regulation of the cGMP level and the recovery of the nerve impulse response after a conditioning stimulus were impaired in isolated antennae. This correlation might indicate a physiological role for cGMP in the setting of the parameters of the nerve impulse response.