Susan A. Levine

MOLECULAR CLONING AND EXPRESSION OF A CDNA ENCODING THE RABBIT ILEAL VILLUS CELL BASOLATERAL MEMBRANE NA+/H+ EXCHANGER

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5′‐untranslated region, 2451 bp of open reading frame and 1163 bp of 3′‐untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are conserved in this protein, suggesting that it is a glycoprotein. Stable transfection of the cDNA into an Na+/H+ exchanger deficient fibroblast cell line, established Na+/H+ exchange. The Na+/H+ exchanger was stimulated by serum and a phorbol ester but not by 8‐Br‐cAMP. In Northern blot analysis, the cDNA hybridized to a 4.8 kb message in rabbit ileal villus cells, kidney cortex, kidney medulla, adrenal gland, brain and descending colon and to a 5.2 kb message in cultured human colonic cancer cell lines, HT29‐18 and Caco‐2. In immunoblotting, a polyclonal antibody raised against a fusion protein of beta‐galactosidase and the C‐terminal 158 amino acids of the human Na+/H+ exchanger identified a rabbit ileal basolateral membrane protein of 94 kd and only weakly interacted with the ileal brush border membrane. In immunocytochemical studies using ileal villus and crypt epithelial cells, the same antibody identified basolateral and not brush border epitopes. Restriction analysis of genomic DNA with a 462 bp PstI‐AccI fragment of the rabbit Na+/H+ exchanger strongly suggests the existence of closely related Na+/H+ exchanger genes. The near identity of the basolateral Na+/H+ exchanger and the human Na+/H+ exchanger plus the ubiquitous expression of this message suggests that the ileal basolateral Na+/H+ exchanger is the ‘housekeeping’ Na+/H+ exchanger.

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Abstract.Elevation in intracellular Ca2+ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T84 cells, a human colon cancer line and a model Cl− secretory epithelial cell. The Ca2+ ionophore A23187, which was used to increase the intracellular Ca2+ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na+/mannitol serosal-to-mucosal flux analysis performed across the T84 monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na+ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringers-HCO3 solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca2+; loading the T84 cells with the intracellular Ca2+ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca2+ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca2+/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca2+. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca2+ decreases tight junction resistance in the T84 monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring.

Molecular Properties, Kinetics And Regulation Of Mammalian Na+/H+ Exchangers

Na+/H+exchange was first described by Murer, Hopfer and Kinne [19] in renal brush border membrane vesicles. This process is mediated by Na+/H+exchangers which catalyze the exchange of extracellular Na+for intracellular H with a stoichiometry of 1:1. Na+/H+exchangers have multiple functions, including pH homeostasis, volume regulation, cell proliferation, and transcellular Na+absorption [reviewed in 12]. In no cell is it the only mechanism for any one of these functions. For instance, multiple mechanisms of pH homeostasis are present in most eukaryotic cells including a c┌/HCO3-exchanger, a NaHCO3co-transporter, a Na+- dependent cr/HCO3-exchanger and multiple mechanisms of hT extrusion [reviewed in 15], including the H-K-ATPase pump. In this review, we will focus on recent advances in identification and understanding of the structure/function relationships and acute protein kinase regulation of members of the mammalian Na+/H+exchanger gene family.

Surgical reconstruction of the nasolacrimal system in the horse

Summary Facial trauma resulted in unilateral disruption of the nasolacrimal duct in two horses. The disruption occurred in the bony lacrimal canal adjacent to the maxillary sinus. In each case, the intact proximal part of the nasolacrimal duct was successfully stented into the lumen of the maxillary sinus. either the nasolacrimal opening at the nares or from one of the puncta. A disrupted nasolacrimal duct can heal if it can be stented with a cannula for an extended period of time,6 but in some cases, the disrupted duct cannot be cannulated, and those horses have previously been relegated to potential long term epiphora. This report describes a technique whereby the nasolacrimal system is restored by diversion of the caudal part of the nasolacrimal duct into the maxillary sinus.

Molecular Studies of Members of the Mammalian Na+/H+ Exchanger Gene Family

The brief history of the contribution of molecular biologic studies to the understanding of the Na+/H+ exchanger gene family is not unlike the history of studies of other transport proteins. Many years of results from physiologic and biochemical studies provided the background to allow strategies for the molecular recognition of an initial member of the Na+/H+ exchanger gene family. This was followed by recognition of the existence of a gene family, which even now is only partially defined. Rapid advances followed concerning location, regulation, and structure/function relationships, all of which have served to extend the previous physiologic studies. Current studies involve “torturing” the specific transport proteins by deletion and point mutation and creation of chimeric constructs to further explore structure/function studies. These are descriptive studies that are attempting to gain clues as to how the proteins carry out transport and are regulated. However, they fall short of defining how the proteins work, which presumably will follow from crystallagraphic techniques, although no mammalian transport protein has yet yielded the required information using any approach or combination of approaches.

New drug treatments for diarrhoea

Abstract. This paper reviews the scientific background to the development of new drugs for the treatment of diarrhoeal diseases, and it includes an update of three classes of drugs which may prove useful; gut specific α2‐adrenergic agonists, intestinal Cl− channel blockers, and somatostatin analogues.