Every time we twitch, even the tiniest movement, no matter how strong or weak, is coordinated by complex networks of neurones. But invertebrates orchestrate muscular contractions with a much simpler neural network. They achieve fine control by modulating muscle nerve signals. Sabine Kreissl from the University of Konstanz in Germany explains that one signal-modulating molecule, the neuropeptide proctolin, modulates many neuromuscular processes in the isopod Idotea emarginata. Released from neurones, proctolin binds receptors on muscle cells, and enhances depolarisation induced muscle contraction. But `coupling the receptor to its target in the cell is a black box' explains Kreissl. Curious to discover the muscle cell signalling cascade that is triggered by the neuropeptide proctolin, Kreissl and her colleagues Berit Philipp and Nicole Rogalla began investigating the contents of proctolin's black box (p. 531).

Knowing that proctolin's effects could be simulated by stimulating components of the cAMP signalling pathway, Philipp began investigating cAMP levels in muscle cells to see if a cAMP dependent pathway mediated proctolin's effect. But when she measured the levels of cAMP in muscle cells, they failed to respond to proctolin. Next, Rogalla tested whether cAMP might regulate muscle contraction. She inhibited a key component of the cAMP signalling pathway and measuring the degree to which the contraction was enhanced. If a cAMP pathway regulated the contraction, she expected the contraction to become weaker; but the contraction strength was unaffected. cAMP did not play a role in transmitting the proctolin signal. Some other signalling cascade must mediate the neuropetide's effects.

Turning to the literature, Kreissl realised that another signalling molecule could be involved in modulating muscle contractions; cGMP. Knowing that cGMP stimulates muscle relaxation in smooth muscle, Kreissl suspected that falling levels of the signalling molecule could in turn cause muscle contraction. Following Kreissl's hunch, Philipp measured the levels of cGMP in the isopod's muscle cells after exposure to proctolin, and found that the signalling molecule's level fell. And when Rogalla investigated cGMP's effect on the strength of the muscle cell's contraction by augmenting its cGMP levels, she found the strength of contraction diminished. cGMP must play a role in proctolin signalling. `cGMP had never been implicated before in proctolin signalling' Kreissl says but it seemed as if it may hold the key to proctolin's black box. However she still didn't know which signalling pathway linked proctolin to cGMP.

Back in the library, the team discovered that protein kinase C (PKC) was a component of the cGMP signalling pathway in vertebrates. Knowing that a PKC pathway had already been implicated by other scientists in proctolin signalling, Kreissl decided to inhibit the protein to see if this was the pathway that mediated proctolin's muscle cell effects. Measuring the muscle cell contraction while applying the protein kinase C inhibitor, Rogalla saw the contraction decrease, and when she supplemented the inhibited cell's cGMP supply with an artificial analogue, she saw the contraction gain force again. Proctolin was mediating its effect in the muscle through a protein kinase C dependent cGMP pathway.

Philipp, B., Rogalla, N. and Kreissl, S.(
2006
). The neuropeptide proctolin potentiates contractions and reduces cGMP concentration via a PKC-dependent pathway.
J. Exp. Biol.
209
,
531
-540.