EPITHELIAL TRANSPORT LABORATORY

David Ian Cook

  

  

RESEARCH

PERSONNEL in 1994 and 1995

Dr David Ian Cook Associate Professor University 1986-
Dr John Atherton Young Professor, Dean
and Pro-Vice-Chancellor
University 1966-
Dr Philip Poronnik Senior Research Officer ARC 1987-
Dr Permsak Komwatana Postdoctoral Fellow Faculty of Medicine 1993-
Dr Tsunetoshi Hayashi Visiting scholar 1993-95
Dr Margot L. Day Postdoctoral Fellow NHMRC 1993-
Dr Anuwat Dinudom Postdoctoral Fellow Medical Foundation 1994-
Francis J.W. Lee Technical Officer 1988-
Carolyn A. Gibb PhD student ARC 1993-
Anthony Weinhaus PhD student (0.5) OPRS 1990-94
Abigail G. Widin PhD student USCRF 1995-
David R. Ireland BMedSc(Hons) student 1994
Michael J. Watson BMedSc(Hons) student 1994

Current effective full-time personnel = 9.0

  
This Laboratory investigates the transport processes in the cell membrane and the manner in which cells control membrane transport activity.
  

PROJECTS in 1994

Ion transport in early embryonic development

Margot Day, Carolyn Gibb, Abigail Widin

During 1994, four aspects of ion transport were investigated in mouse early embryos.

First, studies on K+ channels in early embryonic development were continued. It had previously been shown that there is a 240 pS K+ channel in early embryos which is controlled by the cell cycle and that the cyclical changes in the activity of this channel are not dependent on protein synthesis. In 1994, it was shown that cytochalasin D which disrupts the actin cyto-skeleton does not affect the cyclic inactivation and re-activation of the channel. Thus the channel is not being controlled by the actin cytoskeleton. Studies were also conducted on 1-cell embryos which had been bisected so that one half contained both pro-nuclei and the other half contained no pro-nuclei. From these studies, it became clear that the regulation of the 240 pS channel does not depend on the presence of a nucleus. This confirmed the Lab's earlier studies which indicated that the cyclical changes in activity of the 240 pS K+ channel are attributable to a previously unsuspected cytosolic cell cycle clock.

Second, the Lab extended its search for compounds that block the 240 pS K+ channel. It was found that the channel is blocked by low concentrations of the compound quinacrine. This was not related to the anti-malarial activity of quinacrine, as other potent anti-malarials such as quinidine and mefloquin, block the channel poorly, if at all. It was also not related to the known effects of quinacrine as a DNA intercalator, an inhibitor of phospho-lipase A2 or an inhibitor of calmodulin, since structurally unrelated compounds that share these actions with quinacrine did not affect the channel. Further, it was found that quinacrine was highly effective in blocking cell division. This effect was not shared by inhibitors of phospholipase A2 nor by inhibitors of calmodulin. DNA intercalators such as mitomycin did block cell division, but only at earlier stages of the cell cycle than quinacrine. These data suggested that the 240 pS K+ channel may have some role in controlling cell division.

Third, studies were performed on the effects of the cell cycle on the magnitude of the T-type Ca2+ current. It was found that the T-current declined during the first cell cycle reaching a minimum during the first mitosis. Its magnitude then increased following the first cleavage, before declining again through the second cell cycle. These changes in the amplitude of the T-current were out-of-phase with the changes in the activity of 240 pS K+ channel during the cell cycle, but otherwise appeared to be driven by the same cytosolic cell cycle clock.

Fourth, studies were continued on the control of pH in 2-cell mouse embryos. In 1994 the lactate transport system and the Na+-H+ exchanger in these cells were characterized. No evidence could be found of a H+ conductance, suggesting that earlier reports that these cells do contain one is probably attributable to failure to take into account the influence of the lactate transport system.

Ion transport mechanisms in sheep parotid cells

Phillip Poronnik, Toshi Hayashi, Michael Watson

In 1994, studies were extended on the acetylcholine-activated K+ and anion channels in sheep parotid cells. The Lab confirmed its preliminary studies indicating that the acetylcholine-activated K+ current had a sensitivity pattern to K+ channel blockers, such as tetraethylammonium, which was quite different to that characteristic of voltage- and Ca+-activated K+ (BK) channels. The Lab also used optical measurements of the volume of isolated sheep secretory endpieces to show that acetylcholine-activated K+ efflux from these cells has a sensitivity to blockers similar to that observed for the acetylcholine-activated K+ current. Thus in sheep parotid secretory cells, the acetylcholine-activated K+ current is not carried by BK channels.

The findings in sheep parotid cells conflicted with published reports that in mouse mandibular secretory cells, BK channels do carry the acetylcholine-activated current. Using patch-clamp and microspectrofluorimetric methods, as well as optical measurement of cell volume, it was shown that in mouse mandibular secretory ducts as is the case in the sheep parotid, BK channels do not carry the acetylcholine-activated current. Finally, the K+ channel type activated by acetylcholine was found to be a 50 pS channel, which is not sensitive to tetraethylammonium and which is not voltage-activated.

Studies on isolated intralobular ducts

Anuwat Dinudom, Persak Komwatana, David Ireland

During 1994, studies were continued using whole-cell patch-clamp techniques on the Cl- currents, the K+ currents and the Na+ currents in these cells.

In studies on the Cl- conductances in mouse intralobular duct cells, progress was made in two areas. First, the cyclic AMP-activated Cl- conductance in mouse intralobular duct cells was found to have an ion selectivity pattern, a blocker sensitivity pattern and a voltage-sensitivity pattern characteristic those of the Cl- conductance associated with the cystic fibrosis transmembrane regulator protein (CFTR). Second, it was established that the hyperpolarization-activated Cl- current in these cells was inhibited both by increasing and by decreasing the osmolality of the bath solution. This behaviour distinguished it from the ClC-2 Cl- conductance reported in other tissues, although the similarity in the biophysical properties of these two conductances suggests they must be closely related.

In studies on the Na+ conductance in mouse mandibular intralobular duct cells, progress was made in two areas. First, the Lab developed a new technique of fluctuation ensemble analysis which permitted determination of the single channel conductance of the amiloride- sensitive Na+ channels from a whole-cell recording. Using this method, the Lab established that the saturation of the whole-cell Na+ current with increasing extracellular Na+ concentration was attributable to saturation of the single channel conductance, not to inactivation of the channels. The reduction in the whole-cell Na+ current with increasing intracellular Na+ was, on the other hand, due to inactivation of the Na+ channels. Second, it was established that the anion composition of the cytosol of intralobular duct cells regulates the Na+ conductance by altering the activity of a G protein, which acts to inhibit the Na+ channels.

Studies on the K+ conductance in mouse mandibular intralobular ducts cells indicated that it was predominantly an ATP-sensitive K+ conductance.
  

RESEARCH PLANNED for 1995

During 1995, the Lab will continue working in all three major areas of interest. The work on early embryos will concentrate on establishing the changes in pH regulating mechanisms which take place in normal embryonic development, and on defining the intracellular mechanisms by which the activity of the 240 pS K+ channel is controlled. The work on salivary secretion in the sheep parotid will concentrate on defining the anion conductance which is activated by acetylcholine in sheep parotid cells and on further establishing the characteristics of the acetylcholine-activated K+ current in mouse mandibular cells. Finally, the work on intralobular ducts will include microspectrofluorimetric measurements to define the mechanisms by which duct cells control their pH as well as further patch-clamp studies on the mechanisms by which the amiloride-sensitive Na+ channels are controlled.
  

COLLABORATIONS

Ion channels in mouse early embryos:
Prof. Martin Johnson, Dept of Anatomy, Univ. of Cambridge, U.K. (1989-present).

Control of insulin secretion in foetal pancreatic B cells:
Dr Bernard Tuch, Prince of Wales Hospital (1991-1995).

Control of cytosolic pH in sertoli cells:
Dr Eleonora Gorczynska, Dept of Obstetrics and Gynaecology (from 1995).
  

THESES PASSED in 1994

BMedSc(Hons)

Ireland D (1994) A patch-clamp study of regulation of ion transport in mouse mandibular intralobular duct cells (Result: 1st Class)

Watson M (1994) Volumetric studies on sheep parotid and mouse mandibular endpieces (Result: 2nd Class, Div. 1)

Widin A (1994) The role of potassium and calcium channels in mouse early embryos (Result: 2nd Class, Div. 1)
  

OTHER RESEARCH ACTIVITIES in 1994

Appointments held

Sub-Dean (Research).

University committees

Member, Research Policy Committee.

Member, Research Forum.

Member, Postgraduate Awards Committee.

Member, University Research Grants Committee.

Faculty committees

Member, Research Committee.

Chair, Resources Allocation Standing Committee.

Member, Equipment Advisory Committee.

Refereeing

Manuscripts:
for Pflugers Archiv European Journal of Physiology (6), Journal of Physiology (1), Cell Calcium (1), Journal of Membrane Biology (1).

Grant applications:
for NHMRC (3), ARC (3), NHF (2), St Vincent's Hospital (1), MSD Research Foundation (1).

International conferences

Invited speaker, 8th International Symposium on Ruminant Physiology, Willingen, Germany, (Sep).

Invited speaker, 3rd Congress of the Federation of Asian and Oceanian Physiological Societies, Shanghai, China (Nov).

Invited speaker, Joint Symposium, of the Hong Kong Physiological and Pharmacological Societies, on Epithelial Transport, Hong Kong (Nov).

National conference

Symposium Speaker, Annual Meeting of the Australian Neuroscience Society, Sydney (Jan).

Seminars

Overseas

Dept of Physiology, Catholic Univ. of Leuven, Belgium (Sep).

Depts of Physiology at the Universities of Freiburg, Frankfurt and Wurzburg, Germany (Oct).

Local

Division of Neuroscience, John Curtin School of Medical Research, Canberra (Jul).
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): Cook: 92; Young: 197

  

FUNDING in 1994 and 1995

ARC The mechanism of secretion by
sheep parotid glands
Cook DI 1993
1994 $55,500
1995 $56,763

ARC The mechanisms of secretion by
rat pancreatic acinar cells
Cook DI 1993
1994 $20,000
1995 $20,000

NHMRC Role of membrane transport systems in
early embryonic development
Cook DI 1995 $60,983
1996
1997

NHMRC Studies on electroneutral transport in
salivary duct cells
Cook DI 1994 $45,525
Young JA 1995 $46,152
1996

NHMRC Studies on the role of ion channels in
early embryonic development
Cook DI 1992
1993
1994 $41,537

NHMRC Microfluorimetric imaging equipment Cook DI 1995 $33,713

UEG Fluorescence imaging equipment Cook DI 1994 $46,000

USCRF Role of K+ channels in the cell cycle Cook DI 1995 $23,000
1996

Ramaciotti Single channel studies on Na+ and Cl-
channels in the apical membrane of
mouse mandibular salivary ducts
Dinudom A 1995 $15,000

Australian
Kidney Fdn
Mechanisms by which Na+ absorption
is controlled
Cook DI 1995 $7,500

Total for 1994: $208,562 + share of confocal equipment grants

Total for 1995: $263,021

  

  

TEACHING

COURSES TAUGHT

Medicine 1

Lectures: 16, on introductory cell biology.

Tutorials: 12.

Examination: 50 min of true-false and short answer questions.

Medicine 2

Lectures: 3, on cystic fibrosis.

Examination: 2,000 word essay.

Medical Science 2: Human Life Sciences 2

Lectures: 4, on epithelial physiology.

Practicals: 4, each of 4 h, on permeability.

Tutorials: 4, each of 1 h.

Examination: 30 min of multiple choice questions.

Human Life Sciences 3

Lectures: 13 on cell physiology.

Group sessions: 18, each of 1 h.

Assessment: 1 essay of 2,000 words, 1 report of 300 words.

POSTGRADUATE TEACHING

18 h of lectures on cellular, renal, gastrointestinal and endocrine physiology in the Part 1 course of the Faculty of Anaesthetists.
  

TEACHING HOURS in 1994

Med 1 Med 2 HLS 2 HLS 3 Total
Lectures 16 3 4 13 35
Practicals (no.) - - 16 (4) - 16
Tutorials 12 - 4 18 34

Total formal contact teaching time = 85 h

  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

Faculty of Medicine committees

Member, Medicine 1 Board of Examiners.

Member, Medicine 2 Board of Examiners.

Chair, Years 1 and 2 Curriculum Committee.

Member, Faculty Planning Advisory Committee.

Chair, Resource Allocation Sub-Committee.

Member, Research Committee.

Member, Equipment Advisory Committee.

Member, Admissions Committee.

Member, Working Parties on teaching of Cells and Molecules, Renal Medicine and Gastroenterology and Nutrition in the graduate degree.

Service to the Department

Academic-in-charge, Medicine 1.

Academic-in-charge, Medicine 2.

Academic-in-charge, Human Life Sciences 3 (core-course).