The regulation of ion transport in the midgut of the lepidopteran larva Manduca sexta

Sumner, John-Paul (1995) The regulation of ion transport in the midgut of the lepidopteran larva Manduca sexta. PhD thesis, University of Glasgow.

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Rapid growth during the larval stage of Lepidopteran insects is facilitated by vigorous transepithelial K+ transport from blood to gut lumen across the midgut. Active transport of K+ into the midgut lumen is required to energise amino acid uptake and is believed to contribute to the generation of the extremely high pH found in insect midgut. K+ transport is facilitated by a V-ATPase and K+/nH+ antiporter on the apical plasma membrane of the midgut goblet cell. A transmembrane voltage is generated by V-ATPase driven proton transport, consequently potassium is secreted by exchange for protons via the antiporter. This thesis confirms that the K+ transport system is abolished during periods of non feeding prior to pupation and during larval/larval moults. The moult from fourth to fifth instar was studied in detail. Transepithelial voltage, indicating net active K+ transport, was found to be approximately 100 mV during feeding periods but was found to fall to 0 mV during the moult. The transepithelial voltage was regenerated upon exit from the moult during ecdysis, just prior to resumption of feeding behaviour. The short circuit current was found to mirror these results. The pH of the midgut lumen was found to decline over the period of K+ transport inactivation. Thus during a moult, when the midgut is void of food, the K+ transport system is apparently not required and is consequently switched off. A transmembrane voltage is regenerated prior to the next gorge of food. The identity of the regulatory component of the K+ transport system was sought. The K+ pump is composed of two main components: the V-ATPase and the antiporter. ATPase activity assays on partially purified goblet cell apical membranes (GCAM) demonstrated that the V-ATPase was inactivated during the moult. ATP dependent proton transport into GCAM derived vesicles was also inactivated during the moult. ATP independent (antiporter) activity was not inhibited in vesicles derived from GCAM during the moult. The V-ATPase component thus appears to be the target of a control mechanism. In an effort to elucidate the mechanism of inactivation of the V-ATPase the V-ATPase structure was investigated using SDS gel electrophoresis. GCAM membranes, extracted from moulting or feeding larvae, were run on SDS gels and the V-ATPase subunit components were compared. Loss of V-ATPase activity paralleled the disappearance of specific V-ATPase subunits. The subunits missing were those considered to compose the peripheral V1 catalytic 'head' of the V-ATPase molecule. The integral membrane Vo subunits remained in the GCAM of moulting larvae. A mechanism, believed to be the first demonstration of which in vivo, of inactivation of a V-ATPase by dissociation of V1 from Vo is discussed. An attempt was made to reproduce the hormonal and intra cellular signals required for regulation of V-ATPase activity in vitro. cGMP was found to modestly activate transepithelial K+ transport. NADPH diaphorase staining suggested that nitric oxide synthase was present in the midgut tissue. However, sodium nitroprusside (a spontaneous generator of nitric oxide) did not stimulate K+ transport. This thesis has identified that K+ transport is regulated and that regulation occurs at the level of the V-ATPase. The V-ATPase is inactivated by the loss of the V1 domain of the molecule. Transport may be manipulated in vitro by various secondary messengers.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Physiology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Dow, Dr. Julian
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71809
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 17 Jun 2021 13:37
Thesis DOI: 10.5525/gla.thesis.71809

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