Electrogenic Ion Transport in the Midgut of the Lepidopteran Larva, Manduca sexta (Tobacco Hornworm)

Peacock, Jillian Mary (1991) Electrogenic Ion Transport in the Midgut of the Lepidopteran Larva, Manduca sexta (Tobacco Hornworm). PhD thesis, University of Glasgow.

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The midgut of Manduca sexta and other Lepidopteran larval types has a large electrical gradient approaching 150mV (lumen-side positive) or over 1 mA cm-2 in vivo. This gradient is generated by electrogenic transport of K+ from the blood to the midgut lumen via a unique K+ ATPase located on the goblet cell apical membrane. Active transport of K+ occurs in all three regions of the midgut, however morphological and functional differences result in generation of a high luminal pH of 11-12 only in the anterior and middle regions, with a return to dietary pH levels in the posterior region. Microelectrode impalements of the goblet cell cavity and other midgut cellular compartments has shown that the electrical potential difference across the goblet cell apical membrane in vivo is 269mV which is sufficient to explain a pH of 11.6 in the cavity, using Nernstian dynamics. This evidence supports a primary role for the K+ ATPase of providing the electrical gradient required for high pH generation. The advantages of having high luminal alkalinity for the insect larva are several; (1) Digestive hydrolysis may be accelerated resulting in faster growth and more efficient utilisation of dietary energy. (2) Limitation of gut living pathogenic microorganisms which normally prefer an acidic environment. (3) Protection from dietary tannins which bind to proteins at low pH and therefore reduce digestibility. The presence of high [K+] in the diet led to the original theory that K+ was actively excreted to prevent toxic accumulation in the haemolymph; however, now, there is evidence to suggest that excretion is not the primary role of the K+ ATPase as; (1) Some phytophagous insect larvae do not possess goblet cells and therefore do not actively excrete K+ via cavity-located ATPases, (2) The presence of a complex apical valve at the mouth of the cavity would restrict, rather than facilitate K+ excretion, (3) The electrical gradient produced by active K+ transport across goblet cells actually drives uptake of K+ into the blood via an amino acid cotransport mechanism across columnar cell apical membranes, making a role in excretion seem irrational, (4) K+ activity of the diet is not overly high, however Na+ is low resulting in a larger than normal K: Na ratio. The theories supporting active K+ transport in Lepidopteran midgut have recently been challenged. Some workers believe the pump to be an electrogenic H+ATPase which drives an electroneutral, or electroresponsive K+/H+ exchange. This model explains the electrical potential difference and the transport of K+ into the lumen, but it is difficult to reconcile with the high pH found in the midgut lumen, and the estimated pH of 11.6 thought to exist in the cavity following determination of the goblet cell apical membrane potential. The work carried out during the course of this project attempted to resolve the discrepancies in models for active ion transport using electrophysiological, membrane biochemical and culture techniques. Microelectrode impalements of three midgut cellular compartments yielded the value of 269mV for the goblet cavity membrane mentioned above. In addition, these compartments were probed with a pH sensitive fluorescent dye, acridine orange, whilst monitoring transepithelial potential. When the pump was active (high TEP), no dye was found in the cavities, indicating alkalinity: However, when the pump was inactive (low TEP), for instance in the absence of K+ from the bathing solution, cavity acidification occurred. Furthermore, permeabilization of isolated midgut, using steroid glycosides, abolished the TEP of isolated midgut. Subsequent addition of ATP induced a transient rise of TEP. These results support the hypothesis that an active K+ ATPase located on the goblet cell apical membrane is responsible for generation of the high pH found in the midgut lumen of Manduca sexta larvae. In addition, midgut cells, which were enzymically dissociated from the tissue, were shown to be maintained in culture for more than 2 weeks. This finding, together with the demonstrated lectin-specificity for columnar cells, could be applied in cell and membrane isolation procedures, which in turn would permit more precise biochemical and molecular biological determinations of enzyme characteristics.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Cellular biology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78281
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:34
Last Modified: 30 Jan 2020 15:34
URI: http://theses.gla.ac.uk/id/eprint/78281

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