Biomedical subjects

Gary E Shull

Publications and source records attributed to Gary E Shull.

At least 19 recordsLinked to original sources

PubMed2006

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter.

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Acidosis↗

PubMed2006

PAT-1 (Slc26a6) is the predominant apical membrane Cl-/HCO3- exchanger in the upper villous epithelium of the murine duodenum.

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.

Animals↗

PubMed2006

Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia.

Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na(+)/Ca(2+) exchanger (NCX) contribute to intracellular Na(+) and Ca(2+) overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca(2+) (Ca(m)(2+)) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca(2+) (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca(2+) was reduced by approximately 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca(m)(2+) accumulation. Moreover, OGD/REOX in NKCC1(+/+) astrocytes caused dissipation of the mitochondrial membrane potential (Psi(m)) to 42 +/- 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of Psi(m) was attenuated significantly (66 +/- 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca(2+) that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca(m)(2+) was accompanied by loss of Psi(m) and cytochrome c release in NKCC1(+/+) astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca(m)(2+) homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia.

Animals↗

PubMed2006

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice.

We previously showed that plasma membrane Ca(2+)-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca(2+) ([Ca(2+)](i)) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1(+/-), Pmca4(+/-), Pmca4(-/-), and Pmca1(+/-)Pmca4(-/-) mice. There were no differences in basal [Ca(2+)](i) values between bladder preparations. KCl (80 mM) elicited both larger forces (150-190%) and increases in [Ca(2+)](i) (130-180%) in smooth muscle from Pmca1(+/-) and Pmca1(+/-)Pmca4(-/-) bladders than those in WT or Pmca4(-/-). The responses to carbachol (CCh: 10 muM) were also greater in Pmca1(+/-) (120-150%) than in WT bladders. In contrast, the responses in Pmca4(-/-) and Pmca1(+/-)Pmca4(-/-) bladders to CCh were significantly smaller (40-50%) than WT. The rise in half-times of force and [Ca(2+)](i) increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4(-/-) (130-190%) and Pmca1(+/-)Pmca4(-/-) (120-250%) bladders, but not in Pmca1(+/-) bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca(2+) clearance, while PMCA4 is essential for the [Ca(2+)](i) increase and contractile response to the CCh receptor-mediated signal transduction pathway.

Animals↗

PubMed2006

Ouabain inhibits tubuloglomerular feedback in mutant mice with ouabain-sensitive alpha1 Na,K-ATPase.

Initiation of tubuloglomerular feedback (TGF) depends on Na-K-2Cl co-transport in the macula densa (MD), but it is less clear whether Na,K-ATPase is responsible for establishing the inward Na+ gradient. It has been proposed that apical colonic H,K-ATPase, perhaps in concert with the Na/H exchanger (NHE2), may account for MD Na+ exit in these cells. This study evaluated TGF responses by micropuncture in mutant mice with altered ouabain sensitivity of the alpha1 and alpha2 Na,K-ATPase isoforms. TGF responses in alpha1-sensitive/alpha2-resistant mice were inhibited by intravenous ouabain (control stop-flow pressure = 9.7 +/- 0.9 versus 1.6 +/- 0.5 mmHg with intravenous ouabain). Subsequent inclusion of cyclohexyladenosine (10 microM) in the tubule perfusate confirmed the ability of the afferent arteriole to contract in the presence of ouabain. In alpha1-resistant/alpha2-resistant mice, ouabain infusion had no effect on TGF responses. In separate experiments, loop of Henle perfusion with 50 microM ouabain decreased TGF responses (control stop-flow pressure) from 10.5 +/- 1.1 to 3.9 +/- 1.0 mmHg in alpha1-sensitive/alpha2-resistant mice but had no effect in alpha1-resistant/alpha2-resistant mice, and afferent arteriole responsiveness again was confirmed by cyclohexyladenosine. TGF responses in NHE2 and colonic H,K-ATPase knockout mice were not different from those of wild-type mice. These data indicate that TGF requires activity of the alpha1 Na,K-ATPase, presumably in the MD. Furthermore, the data show that neither NHE2 nor colonic H,K-ATPase is essential for initiation of TGF responses.

Adenosine↗

PubMed2006

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice.

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.

Acids↗

PubMed2006

Loss of acidification of anterior prostate fluids in Atp12a-null mutant mice indicates that nongastric H-K-ATPase functions as proton pump in vivo.

The physiological functions of nongastric (colonic) H-K-ATPase (gene symbol Atp12a), unlike those of Na-K-ATPase and gastric H-K-ATPase, are poorly understood. It has been suggested that it pumps Na+ more efficiently than H+; however, so far, there is no direct evidence that it pumps H+ in vivo. Previously, we found that the nongastric H-K-ATPase alpha-subunit is expressed in apical membranes of rodent anterior prostate epithelium, in a complex with the Na-K-ATPase beta1-subunit. Here we report the effects of Atp12a gene ablation on polarization of the beta1-subunit and secretory function of the anterior prostate. In nongastric H-K-ATPase-deficient prostate, the Na-K-ATPase alpha-subunit resided exclusively in basolateral membranes; however, the beta1-subunit disappeared from apical membranes, demonstrating that beta1 is an authentic subunit of nongastric H-K-ATPase in vivo and that apical localization of beta1 in the prostate is completely dependent on its association with the nongastric H-K-ATPase alpha-subunit. A remarkable reduction in acidification of anterior prostate fluids was observed: pH 6.38 +/- 0.14 for wild-type mice and 6.96 +/- 0.10 for homozygous mutants. These results show that nongastric H-K-ATPase is required for acidification of luminal prostate fluids, thereby providing a strong in vivo correlate of previous functional expression studies demonstrating that it operates as a proton pump.

Animals↗

PubMed2005

Decreased neuronal death in Na+/H+ exchanger isoform 1-null mice after in vitro and in vivo ischemia.

Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism after intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to the disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Three hours of oxygen and glucose deprivation (OGD) followed by 21 h of reoxygenation (REOX) led to 68 +/- 10% cell death. Inhibition of NHE1 with the potent inhibitor cariporide (HOE 642) or genetic ablation of NHE1 reduced OGD-induced cell death by approximately 40-50% (p < 0.05). In NHE1(+/+) neurons, OGD caused a twofold increase in [Na+]i, and 60 min REOX triggered a sevenfold increase. Genetic ablation of NHE1 or HOE 642 treatment had no effects on the OGD-mediated initial Na+(i) rise but reduced the second phase of Na+(i) rise by approximately 40-50%. In addition, 60 min REOX evoked a 1.5-fold increase in [Ca2+]i in NHE1(+/+) neurons, which was abolished by inhibition of either NHE1 or reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX-mediated mitochondrial Ca2+ accumulation and cytochrome c release were attenuated by inhibition of NHE1 activity. In an in vivo focal ischemic model, 2 h of left middle cerebral artery occlusion followed by 24 h of reperfusion induced 84.8 +/- 8.0 mm3 infarction in NHE1(+/+) mice. NHE1(+/+) mice treated with HOE 642 or NHE1 heterozygous mice exhibited a approximately 33% decrease in infarct size (p < 0.05). These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage.

Animals↗

PubMed2005

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice.

We investigated the roles and relationships of plasma membrane Ca(2+)-ATPase (PMCA), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2, and Na(+)/Ca(2+) exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4(-/-)) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1(+/-)Pmca4(-/-)) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca(2+) transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca(2+)-free physiological salt solution containing EGTA. We measured the contributions of Ca(2+) clearance components by inhibiting SERCA2 (with 10 microM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 microM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be approximately 25-30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca(2+) extrusion-relaxation relationship, i.e., Ca(2+) homeostasis, of bladder smooth muscle.

Animals↗

PubMed2005

Haploinsufficiency of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, predisposes mice to squamous cell tumors via a novel mode of cancer susceptibility.

A null mutation in one copy of the Atp2a2 or ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), leads to squamous cell tumors in mice and to Darier disease in humans, a skin disorder that also involves keratinocytes. Here, we examined the time course and genetic mechanisms of tumor development in the mutant animals. Atp2a2+/- mice overexpressed keratins associated with keratinocyte hyperactivation in normal forestomachs as early as 2 months of age. By the age of 5 to 7 months, 22% of mutants had developed papillomas of the forestomach, and 89% of mutants older than 14 months had developed squamous cell papillomas and/or carcinomas, with a preponderance of the latter. Tumors occurred in regions that had keratinized epithelium and were subjected to repeated mechanical irritation. The genetic mechanism of tumorigenesis did not involve loss of heterozygosity, as tumor cells analyzed by laser capture microdissection contained the wild-type Atp2a2 allele. Furthermore, immunoblot and immunohistochemical analysis showed that tumor keratinocytes expressed the SERCA2 protein. Mutations were not observed in the ras proto-oncogenes; however, expression of wild-type ras was up-regulated, with particularly high levels of K-ras. Loss of the p53 tumor suppressor gene occurred in a single massive tumor, whereas other tumors had increased levels of p53 protein but no mutations in the p53 gene. These findings show that SERCA2 haploinsufficiency predisposes mice to tumor development via a novel mode of cancer susceptibility involving a global change in the tumorigenic potential of keratinized epithelium in Atp2a2+/- mice.

Alleles↗

PubMed2005

Hypotension in NKCC1 null mice: role of the kidneys.

NKCC1 null mice are hypotensive, in part, from the absence of NKCC1-mediated vasoconstriction. Whether these mice have renal defects in NaCl and water handling which contribute to the hypotension is unexplored. Therefore, we asked 1) whether NKCC1 (-/-) mice have a defect in the regulation of NaCl and water balance, which might contribute to the observed hypotension and 2) whether the hypotension observed in these mice is accompanied by endocrine abnormalities and/or downregulation of renal Na+ transporter expression. Thus we performed balance studies, semiquantitative immunoblotting, and immunohistochemistry of kidney tissue from NKCC1 (+/+) and NKCC1 (-/-) mice which consumed either a high (2.8% NaCl)- or a low-NaCl (0.01% NaCl) diet for 7 days. Blood pressure was lower in NKCC1 (-/-) than NKCC1 (+/+) mice following either high or low dietary NaCl intake. Relative to wild-type mice, NKCC1 null mice had a lower plasma ANP concentration, a higher plasma renin and a higher serum K+ concentration with inappropriately low urinary K+ excretion, although serum aldosterone was either the same or only slightly increased in the mutant mice. Expression of NHE3, the alpha-subunit of the Na-K-ATPase, NCC, and NKCC2 were higher in NKCC1 null than in wild-type mice, although differences were generally greater during NaCl restriction. NKCC1 null mice had a reduced capacity to excrete free water than wild-type mice, which resulted in hypochloremia following the NaCl-deficient diet. Hypochloremia did not occur from increased aquaporin-1 (AQP1) or 2 protein expression or from redistribution of AQP2 to the apical regions of principal cells. Instead, NKCC1 null mice had a blunted increase in urinary osmolality following vasopressin administration, which should increase free water excretion and attenuate the hypochloremia. In conclusion, aldosterone release is inappropriately low in NKCC1 null mice. Moreover, the action of aldosterone and vasopressin is altered within kidneys of NKCC1 null mice, which likely contributes to their hypotension. Increased Na+ transporter expression, increased plasma renin, and reduced plasma ANP, as observed in NKCC1 null mice, should increase vascular volume and blood pressure, thus minimizing hypotension.

Aldosterone↗

PubMed2005

Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2.

We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na+ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by approximately 75% in NHE1+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 microM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na+ but also Ca2+ homeostasis via reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX led to a 103% increase in intracellular Ca2+ concentration ([Ca2+]i) in NHE1+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca2+]i by 73%. To further investigate changes of Ca2+ signaling, bradykinin-mediated Ca2+ release was evaluated. Bradykinin-mediated intracellular Ca2+ transient in NHE1+/+ astrocytes was increased by approximately 84% after OGD/REOX. However, in NHE1-/- astrocytes or NHE1+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca2+ release was increased by only approximately 34%. Inhibition of the reverse mode of Na+/Ca2+ exchange abolished OGD/REOX-mediated Ca2+ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na+ accumulation subsequently alters Ca2+ homeostasis via Na+/Ca2+ exchange.

Animals↗

PubMed2005

SLC26A9 is expressed in gastric surface epithelial cells, mediates Cl-/HCO3- exchange, and is inhibited by NH4+.

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.

Animals↗

PubMed2005

Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger.

The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.

Achlorhydria↗

PubMed2005

Aberrant retinal projections in congenitally deaf mice: how are phenotypic characteristics specified in development and evolution?

The contribution of sensory input to the formation of sensory system-specific (sensoritopic) connections of the thalamus and midbrain was investigated using mice lacking the Na+-K+-2Cl- cotransporter (NKCC1) or the plasma membrane Ca2+-ATPase isoform2 (PMCA2). Because these mice are congenitally deaf, the developing nervous system has no exposure to sensory-driven neural activity from the auditory system. Here we compared the retinofugal pathway in normal and congenitally deaf mice using intraocular injections of neuroanatomical tracers into each eye, and relating tracer patterns to identified thalamic nuclei and superior colliculus layers. We demonstrate that loss of such activity results in aberrant projections of the retina into nonvisual auditory structures such as the medial geniculate nucleus and the intermediate layers of the superior colliculus. These findings indicate that activity from peripheral sensory receptor arrays is necessary not only for the refinement of developing connections within a unimodal structure, but for the establishment of sensoritopic or sensory-specific connections of unimodal and multimodal structures. We hypothesize that specification of such connections may occur through the modulation of spatial expression patterns of molecules known to be involved in the development of topography of connections between brain structures, such as the ephrins, via activity-dependent, CRE-mediated gene expression.

Animals↗

PubMed2005

Na(+)-dependent chloride transporter (NKCC1)-null mice exhibit less gray and white matter damage after focal cerebral ischemia.

We previously demonstrated that pharmacological inhibition of Na(+)-K(+)-Cl- cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (approximately 85 mm3) in NKCC1 wild-type (NKCC1(+/+)) mice. Infarction volume in NKCC1(-/-) mice was reduced by approximately 30% to 46%. Heterozygous mutant (NKCC1(+/-)) mice showed approximately 28% reduction in infarction (P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1(+/+) mice. In contrast, NKCC1(+/-) and NKCC1(-/-) mice exhibited approximately 50% less edema (P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1(+/+) mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1(-/-) mice exhibited significantly less APP accumulation (P<0.05). Oxygen-glucose deprivation (OGD) induced approximately 67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1(+/+) cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1(-/-) neurons (P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1(+/+) neurons and astrocytes (P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.

Amyloid beta-Protein Precursor↗

PubMed2005

Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(-/-)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors.

BACKGROUND: Gastric neoplasia is common in humans, yet controversy remains over contributions of chronic achlorhydria, gastrinemia and hyperplasia, to cancer risk. To study this, mice lacking the gastric H/K-ATPase (Atp4a(-/-) mice) were used to determine whether chronic loss of acid secretion, with attendant hypergastrinemia, predisposes to cancer phenotype. METHODS: Atp4a(-/-) and Atp4a(+/+) mice, paired for age and gender, were examined at 3, 8, 12 and 20 months for histopathology, and for expression of the trefoil factor family (TFF)1-3, Reg IIIbeta, gamma and delta, osteopontin, CD44, chromogranin A, Crp-ductin, and galectin, all of which are important in cell growth. RESULTS: By 8 months, the glandular stomach of the Atp4a(-/-) mice doubled in weight and thickness, and several modulators of growth were increased. Female Atp4a(-/-) mice were more hyperplastic than Atp4a(-/-) males at 12 and 20 months. By 1 year, severe mucocystic hyperplasia, incomplete intestinal metaplasia, ciliated metaplasia, a shift in mucins from neutral to acidic, and inflammation were widespread. Cells in the mucus pit zone developed a pyloric-type appearance, containing large hyaline-like, periodic acid-Schiff (PAS)-negative/alcian blue-negative inclusions. But critical characteristics of gastric neoplasia, such as nuclear atypia, invasion into the muscularis mucosa, and metastases were absent. In Atp4a(-/-) mice, chromogranin A and histidine decarboxylase, RegIIIgamma and delta, TFF3, osteopontin and CD44 were upregulated while Reg IIIbeta, and TFF1 were reduced. CONCLUSIONS: Chronic achlorhydria and hypergastrinemia in aged Atp4a(-/-) mice produced progressive hyperplasia, mucocystic and incomplete intestinal metaplasia, and the upregulation of growth factors without histological evidence of neoplasia.

Achlorhydria↗

PubMed2004

Deficiency of ATP2C1, a Golgi ion pump, induces secretory pathway defects in endoplasmic reticulum (ER)-associated degradation and sensitivity to ER stress.

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.

Animals↗