Acid and Base Transport

1. Acid and Base Transport

2. Where does it happen? • VERTEBRATES • Stomach - gastric acid secretion • Kidney proximal tubule - urinary acidification (urinary bladder in lower verts) • Exocrine pancreas/duodenum - alkali secretion • Osteoclasts - bone remodeling • INVERTEBRATES • Gut • Excretory Organs

3. Vertebrate stomach - the H+/K+ ATPase mechanism of acid secretion themechanisms of HCl secretion in gastric parietal cells (= oxyntic cells). The K+/H+ ATPase is specifically inhibited by omeprazole, a drug developed to treat peptic ulcers.

4. A complex control system: the vertebrate stomach • Effectors: parietal cells, which secrete 3-4 liters/day of fluid with pH < 1. • 1st order control cells: Enterochromaffin-like (ECL) cells resemble mast cells of the non-specific immune system - secrete the monoamine histamine.

5. 2nd order control: 2 kinds of enteroendocrine cells G cells which secrete the peptide gastrin D cells which secrete the peptide somatostatin

6. The turn-on pathway at the beginning of a meal Amino acids from meal G CELL Bombesin Gastrin CCK-B receptor Ach ECL CELL Histamine Parasympathetic N.S. H2 receptor PARIETAL CELL Norepinephrine (inhibits) Sympathetic N.S. HCl

7. Turn-off pathways that protect the stomach and duodenum Secretin: hormone - from duodenum, secreted when duodenal pH falls D cell somatostatin SST 2 receptor Prostaglandin: from stomach tissues secreted in response to tissue damage ECL cell Histamine Parietal cell HCl

8. Longer term effects of gastrin on ECL cells • 30-120 min: Increase in expression of the gene for histidine decarboxylase – rate-limiting step in histamine synthesis - this gene has Ca++ response elements. • Days-weeks: elevated gastrin levels cause mitosis of ECL cells (hyperplasia) caused by activation of Ca++-sensitive tyrosine kinase

9. Stimulus-secretion coupling in ECL cells • Gastrin binds to CCK-B receptor on ECL cell • CCK-B receptor is coupled to G-protein • G-protein activates Ca++ channels (?) and/or pathway to intracellular Ca++ release- [Ca++] increase is detectable within 30 sec of gastrin binding • rise in Ca++ stimulates exocytosis of histamine-containing vesicles within 5 min.

10. Competition between turn-on and turn-off G proteins for control of Ca++ channels in ECL cells Ca++ CCK-B (gastrin) SSTR 2 (somatostatin) Gq Gi

11. Sites of action of some relevant drugs that, until recently, were used for peptic ulcers • H2 receptor blocker: ranitidine • H+/K+ pump blocker: omeprazole • prostaglandin synthesis blocker: aspirin; can promote peptic ulcers

12. Why are ranitidine and omeprazole now OTC drugs? • Infection with Helicobacter pylori is now recognized as a major causative for peptic ulcers • Such infections are typically acquired through contanimated water and can be treated with antibiotics • Chronic Helicobacter infection also predisposes to stomach cancer

13. The acid-secreting mitochondria-rich cell Key features: The apical membrane contains vacuolar-type H+ ATPase; the basolateral membrane contains Cl-/HCO3- exchanger (AE). The cytoplasm contains abundant carbonic anhydrase and ER vesicles.

14. MR cells from rat kidney (left) and toad urinary bladder (right).

15. Where membrane proteins are in cells: immunohistochemistry. 1. Isolate membrane domain by homogenization/sonication followed by differential centrifugation 2. Solubilize/purify membrane proteins 3. Make monoclonal antibodies to individual membrane proteins

16. 4. Get fluorescent-labeled secondary antibodies. For example, if the monoclonal was raised in mouse cells, get fluorescent-coupled antibodies against the mouse antibody raised in goat, rat, rabbit, etc. 5. Treat tissue samples with the primary antibody and then add the secondary antibody 6. Examine under fluorescence microscope

17. V-Type H+ATPase histochemistry This rat kidney tubule has been stained with antibody to the V-Type H+ ATPase. The stain shows up as yellow-green glow. Only intercalated cells stain. The gaps between stained cells are filled by unstained principal cells, which are not involved in acid-base transport. Some cells have apical labeling only (Alpha cells), some have basolateral stain only (Beta cells) and some have both (Gamma cells).

18. Depending on the homeostatic demand, H+-ATPase and AE can be targeted to apical or basolateral membrane • Turtle bladder and mammalian nephrons can secrete either acid or base • Current evidence suggests that depending on whether the animal is acidotic or alkalotic, V-ATPase and AE containing vesicles may insert in either membrane, allowing the same cell to convert between the A and B cell type.

19. You could read more about this in: Brown, D. & Breton, S. (1996) Mitochondria-rich, proton-secreting epithelial cells J. Exp. Biol. 199, 2345-2358.

20. Bone remodeling and acid secretion • Bone is a complex of Ca Carbonate and Ca Phosphate in an organic matrix, hydroxyapatite. • Bone is deposited by osteoblasts, but it is constantly being remodeled in response to mechanical stress. Bone demolition is the responsibility of osteoclasts.

21. The osteoclast is an honorary A cell The micrograph shows three osteoclasts dissolving a bone surface. The brown color is a deposit of antibody to the V-ATPase. The plasma membrane next to the bone surface could be thought of as an extracellular lysosome.

22. A close encounter of the third kind: an osteoclast landed here. This scanning electron micrograph shows an eroded crater on bone surface resulting from osteoclast activity.

23. Now for a little relief from acid digestion - some alkali-secreting cells.

24. Pancreatic secretion Secretion of alkaline pancreatic juice is turned on as food enters the intestine.The hormone secretin from the duodenum is the signal for this. In A the secretory cells are inactive, with a reserve of vesicles containing transporters. The binding of the hormone with its receptor (B) causes vesicles to fuse with the target membranes, turning on secretion (C).

25. The ‘stomach’ of mosquito larvae Aedes aegypti Overview of Gut Structure

26. Acid-base cycle in midgut • Anterior midgut raises lumen pH to >10 • Posterior midgut returns pH to <8 • Both processes are driven by V-ATPase • V-ATPase is basal in AMG; apical in PMG • Extreme alkalinization apparently protects the animal against bacterial colonization. • Buffered medium can overwhelm the alkali secretion cycle, and the animals die as a result.

27. In anterior stomach, V-ATPase is characteristically concentrated in distinctive patterns at the basal membrane. wholemount sectioned

28. Intracellular pH is measured using the fluorescent probe BCECF A: normal light microscope view B: fluorescence microscope view with areas of interest marked C: 4 representative tissues’ response to serotonin and postexperiment calibration Serotonin-stimulated guts secrete alkali and the cytoplasm also becomes very alkaline.

29. Comparison of simultaneous responses of an anterior midgut ‘area of interest’ compared to a posterior midgut one in the same tissue.

30. Intracellular alkalinization could be needed to get enough carbonate for a carbonate transporter; it also increases the upper limit of luminal alkalinization as determined by the energetics of the V-ATPase. Two hypotheses are presently being tested.

31. After this lecture, you should be able to • Describe two cellular mechanisms for secreting acid, and give specific examples. • Describe a general mechanism for secreting alkali, and give at least one specific example.