Further observations on the behaviour of ouabain‐insensitive sodium efflux towards proctolin in barnacle muscle fibres.

Abstract

SUMMARY1. A further study has been made of the stimulatory action of proctolin on the ouabain-insensitive Na+ efflux in single muscle fibres from the barnacle, Balanus nubilus.2. (i) Strontium (Sr2+) behaves as a substitute for external Ca2+. In this case, however, the response to proctolin fails to decay. (ii) Injection of Sr2+ stimulates the ouabain-insensitive Na+ efflux. This effect is mimicked by injecting Ca2 .3. Depolarization of the fibre membrane with 30 and 100 mM-external K+ augments the response to proctolin.4. Pre-injection of GTP or Gpp(NH)p (sodium 5'-guanylylimidodiphosphate) prevents the response to proctolin from completely decaying.5. Pre-injection of guanine nucleotides in conjunction with membrane depolarization stops the response to proctolin from decaying.6. Measurements of Em before and during treatment with proctolin indicate a prompt but small and reversible fall in the membrane potential.7. (i) The aequorin response of fibres pre-treated with ouabain to proctolin is monophasic or multiphasic, and concentration dependent, the minimal effective concentration being in the nanomolar range.(ii) The duration of these signals is usually less than 5 min; this is about half the time it takes for the stimulated Na+ efflux to reach a maximum. (iii) The aequorin response to proctolin occurs quite often in fibres suspended in nominally Ca2+-free artificial sea water. (iv) Sudden graded elevations in external K+ following complete decay of the aequorin response to proctolin are rapidly followed by stepwise transitory increments in light emission.(v) The aequorin response to 100 mM-external K+ is frequently a triplet. 8. (i) Together, these results are in line with the view that the action of proctolin on the ouabain-insensitive Na+ efflux is the result of a temporary fall in internal pCa and that its point of action is the Ca2+ channel, where a putative G protein in the presence of GTP or Gpp(NH)p is able to maintain constancy of the hormonal effect.(ii). They strengthen the argument that the barnacle muscle fibre as a preparation is especially suitable for studies of this kind.

Proctolin activates an inward current whose voltage dependence is modified by extracellular Ca2+

“…In crayfish skeletal muscle, Bishop et al (199 1) showed that proctolin modulates the voltage-dependent activation of Ca *+ channels, effects that would have been masked by the Cd*+ we used. Finally, in barnacle muscle fibers proctolin activates a ouabain-insensitive Na+ efflux (Bittar and Nwoga, 1989) an effect that would have been masked by the low Na+ influx due to the presence of TTX in our preparations. However, although it is certainly possible that the proctolin target neurons display an additional proctolin response, we saw no indication that any of the pharmacological agents we used to isolate the proctolin current for study influenced the amplitude or the character of the proctolin response.…”

“…Proctolin itself has been proposed to act via a CAMP-dependent mechanism (Jennings et al, 1983;Orchard et al, 1989;Bishop et al, 199 1) and via a G-protein (Bittar and Nwoga, 1989) in other preparations. The slow nature of the proctolin response we have described (see Figs.…”

A study of the ouabain‐insensitive sodium efflux in barnacle muscle fibres using phorbol dibutyrate as a probe.

“…The species of barnacles, the methods of dissection, cannulation, microinjection and counting of 22Na+ activity in the effluent and in the fibre, as well as the measurement of the membrane potential, were essentially the same as those described previously (Bittar & Nwoga, 1989b). Solutions with varying concentrations of Ca2+ were prepared by raising or reducing NaCl in 264 EFFECT OF PHORBOL DIBUTYRATE Na+ EFFLUX osmotically equivalent amounts.…”

“…To prepare solutions of furosemide, piretanide and bumetanide in millimolar concentrations, methanol was used as the solvent and its final concentration in ASW was 1 %. Changes in myoplasmic free Ca2+ were monitored with the Ca2+_ sensitive photoprotein, aequorin, as described by Ashley & Ridgway (1970) in this preparation and by Bittar & Nwoga (1989b). All experiments were performed at room temperature (between 22 and 24 0C).…”

Peptide cotransmitter potentiates calcium channel activity in crayfish skeletal muscle

“…In barnacle muscle fibers, for example, a proctolin response (in this case, a proctolin-stimulated, ouabain-insensitive Na2+ efflux) was found to be eliminated with Cd*+ and Co2+ and correlated with increased cytosolic free CaZ+ (which was measured with aequorin; Bittar and Nwoga, 1989). Similar Ca*+ channel blockers were found to block proctolin-induced contractures in a number of other muscle preparations, including Limuhs heart muscle (Watson and Hoshi, 1985) lobster opener muscle (Schwartz et al, 1980), and cockroach hypemeural muscle (Hertel and Penzlin, 1986).…”