Biomedical subjects

John N Lorenz

Publications and source records attributed to John N Lorenz.

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

Cardiac myosin binding protein C phosphorylation is cardioprotective.

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-C(AllP+)). The allele was bred into a cMyBP-C null background (cMyBP-C((t/t))) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-C(AllP+) was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick-thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-C(AllP+:(t/t)) hearts were resistant to ischemia-reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.

Amino Acid Sequence↗

PubMed2006

Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice.

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P(1-5)) to cardiovascular homeostasis. We used S1P(2) receptor knockout mice (S1P(2)(-/-)) to evaluate the role of S1P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.

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

GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation.

Here we identified growth-differentiation factor 15 (GDF15) (also known as MIC-1), a secreted member of the transforming growth factor (TGF)-beta superfamily, as a novel antihypertrophic regulatory factor in the heart. GDF15 is not expressed in the normal adult heart but is induced in response to conditions that promote hypertrophy and dilated cardiomyopathy. To elucidate the function of GDF15 in the heart, we generated transgenic mice with cardiac-specific overexpression. GDF15 transgenic mice were normal but were partially resistant to pressure overload-induced hypertrophy. Expression of GDF15 in neonatal cardiomyocyte cultures by adenoviral-mediated gene transfer antagonized agonist-induced hypertrophy in vitro. Transient expression of GDF15 outside the heart by intravenous adenoviral delivery, or by direct injection of recombinant GDF15 protein, attenuated ventricular dilation and heart failure in muscle lim protein gene-targeted mice through an endocrine effect. Conversely, examination of Gdf15 gene-targeted mice showed enhanced cardiac hypertrophic growth following pressure overload stimulation. Gdf15 gene-targeted mice also demonstrated a pronounced loss in ventricular performance following only 2 weeks of pressure overload stimulation, whereas wild-type controls maintained function. Mechanistically, GDF15 stimulation promoted activation of SMAD2/3 in cultured neonatal cardiomyocytes. Overexpression of SMAD2 attenuated cardiomyocyte hypertrophy similar to GDF15 treatment, whereas overexpression of the inhibitory SMAD proteins, SMAD6/7, reversed the antihypertrophic effects of GDF15. These results identify GDF15 as a novel autocrine/endocrine factor that antagonizes the hypertrophic response and loss of ventricular performance, possibly through a mechanism involving SMAD proteins.

Animals↗

PubMed2006

The proximal convoluted tubule is a target for the uroguanylin-regulated natriuretic response.

OBJECTIVES AND METHODS: Guanylin and uroguanylin are peptides synthesized in the intestine and kidney that are postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of guanylin and uroguanylin in the regulation of sodium excretion, we used gene-targeted mice in which the uroguanylin, guanylin or the peptide receptor guanylate cyclase C gene expression had been ablated. RESULTS: Metabolic balance studies demonstrated that there was impaired excretion of a sodium load in uroguanylin (but not in guanylin or guanylate cyclase C) knockout mice. Uroguanylin-dependent natriuresis occurred without an increase in circulating prouroguanylin. A distinct morphological phenotype was present in the proximal convoluted tubules of uroguanylin knockout animals after an enteral salt loading. Marked vacuolization of the proximal convoluted tubule epithelial cells was observed by using light and electron microscopy. There was also a change in the distribution of the sodium hydrogen exchanger 3 (NHE3) after an enteral salt loading. In wild-type animals, there was a partial redistribution of NHE3 from the villus fraction to the less accessible submicrovillus membrane compartment, but this effect was less apparent in uroguanylin knockout animals, presumably resulting in greater Na/H exchange. CONCLUSIONS: Together, these findings further establish a role for uroguanylin in fluid homeostasis and support a role for uroguanylin as an integral component of a signaling mechanism that mediates changes in Na excretion in response to an enteral salt loading. Proximal tubular NHE3 activity is a possible target for uroguanylin-mediated changes in Na excretion.

Analysis of Variance↗

PubMed2006

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site.

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.

Animals↗

PubMed2006

Cerebral ischemia-hypoxia induces intravascular coagulation and autophagy.

Hypoxia is a critical factor for cell death or survival in ischemic stroke, but the pathological consequences of combined ischemia-hypoxia are not fully understood. Here we examine this issue using a modified Levine/Vannucci procedure in adult mice that consists of unilateral common carotid artery occlusion and hypoxia with tightly regulated body temperature. At the cellular level, ischemia-hypoxia produced proinflammatory cytokines and simultaneously activated both prosurvival (eg, synthesis of heat shock 70 protein, phosphorylation of ERK and AKT) and proapoptosis signaling pathways (eg, release of cytochrome c and AIF from mitochondria, cleavage of caspase-9 and -8). However, caspase-3 was not activated, and very few cells completed the apoptosis process. Instead, many damaged neurons showed features of autophagic/lysosomal cell death. At the tissue level, ischemia-hypoxia caused persistent cerebral perfusion deficits even after release of the carotid artery occlusion. These changes were associated with both platelet deposition and fibrin accumulation within the cerebral circulation and would be expected to contribute to infarction. Complementary studies in fibrinogen-deficient mice revealed that the absence of fibrin and/or secondary fibrin-mediated inflammatory processes significantly attenuated brain damage. Together, these results suggest that ischemia-hypoxia is a powerful stimulus for spontaneous coagulation leading to reperfusion deficits and autophagic/lysosomal cell death in brain.

Animals↗

PubMed2005

The highly conserved cardiac glycoside binding site of Na,K-ATPase plays a role in blood pressure regulation.

The Na,K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, which is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, it is unknown whether this site has a natural function in vivo. Here we show that this site plays an important role in the regulation of blood pressure, and it specifically mediates adrenocorticotropic hormone (ACTH)-induced hypertension in mice. We used genetically engineered mice in which the Na,K-ATPase alpha2 isoform, which is normally sensitive to cardiac glycosides, was made resistant to these compounds. Chronic administration of ACTH caused hypertension in WT mice but not in mice with an ouabain-resistant alpha2 isoform of Na,K-ATPase. This finding demonstrates that the cardiac glycoside binding site of the Na,K-ATPase plays an important role in blood pressure regulation, most likely by responding to a naturally occurring ligand. Because the alpha1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant alpha1 isoform was made ouabain-sensitive. Mice with the ouabain-sensitive "human-like" alpha1 isoform and an ouabain-resistant alpha2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the alpha1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the alpha isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides.

Adrenocorticotropic Hormone↗

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

Contribution of the basolateral isoform of the Na-K-2Cl- cotransporter (NKCC1/BSC2) to renin secretion.

Acute administration of loop diuretics like furosemide leads to a stimulation of renin secretion, an effect thought to result from inhibition of Na-K-2Cl cotransporter (NKCC2)-mediated salt transport at the luminal surface of the macula densa (MD). However, loop diuretics also inhibit NKCC1, the second isoform of the Na-K-2Cl cotransporter, with similar potency. In the present study, we examined the influence of furosemide on renin secretion in NKCC1-deficient mice to distinguish between effects of the loop diuretic involving NKCC2 and, by implication, the MD pathway, and effects that might occur via inhibition of NKCC1. Baseline plasma renin concentration (PRC) was 1,212 +/- 211 in NKCC1+/+ (n = 13) and 3,851 +/- 579 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice (n = 14; P = 0.00024). Acute administration of furosemide (50 mg/kg i.p.) increased PRC significantly to 9,324 +/- 1,018 ng ANG I.ml(-1).h(-1) in NKCC1+/+ (n = 13; P < 0.0001 compared with basal) and to 14,188 +/- 2,274 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice [n = 14; P = 0.0002 compared with basal; P = 0.034 compared with wild-type (WT) plus furosemide]. Renin mRNA expression was about threefold higher in NKCC1-/- compared with WT mice. There was considerable recruitment of granular cells to upstream regions of afferent arterioles in NKCC1-/- mice. Patch-clamp studies in single juxtaglomerular granular (JG) cells from WT mice showed an approximately 10% increase in membrane capacitance during incubation with furosemide (10(-4) M), indicating a direct effect of the loop diuretic on renin secretion. No effect of furosemide on membrane capacitance was observed in JG cells from NKCC1-deficient mice. Furosemide (10(-3) M) significantly stimulated renin release from primary cultures of JG cells from WT mice, whereas no response was observed in NKCC1-/- mice. Our data suggest that a functional NKCC1 suppresses basal renin release, at least in part, through a direct effect on JG cells.

Animals↗

PubMed2005

Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase 1.

Abnormal calcium cycling, characteristic of experimental and human heart failure, is associated with impaired sarcoplasmic reticulum calcium uptake activity. This reflects decreases in the cAMP-pathway signaling and increases in type 1 phosphatase activity. The increased protein phosphatase 1 activity is partially due to dephosphorylation and inactivation of its inhibitor-1, promoting dephosphorylation of phospholamban and inhibition of the sarcoplasmic reticulum calcium-pump. Indeed, cardiac-specific expression of a constitutively active inhibitor-1 results in selective enhancement of phospholamban phosphorylation and augmented cardiac contractility at the cellular and intact animal levels. Furthermore, the beta-adrenergic response is enhanced in the transgenic hearts compared with wild types. On aortic constriction, the hypercontractile cardiac function is maintained, hypertrophy is attenuated and there is no decompensation in the transgenics compared with wild-type controls. Notably, acute adenoviral gene delivery of the active inhibitor-1, completely restores function and partially reverses remodeling, including normalization of the hyperactivated p38, in the setting of pre-existing heart failure. Thus, the inhibitor 1 of the type 1 phosphatase may represent an attractive new therapeutic target.

Animals↗

PubMed2005

Genetic profiling reveals global changes in multiple biological pathways in the hearts of Na, K-ATPase alpha 1 isoform haploinsufficient mice.

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The alpha1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the alpha1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the alpha1 heterozygous mice was unexpected since mice heterozygous for the alpha2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from alpha1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the alpha1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by alpha1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase alpha1 isoform activity may be required in numerous cellular processes.

Aldosterone↗

PubMed2004

Renal and intestinal transport defects in Slc26a6-null mice.

SLC26A6 (PAT1, CFEX) is an anion exchanger that is expressed on the apical membrane of the kidney proximal tubule and the small intestine. Modes of transport mediated by SLC26A6 include Cl-/formate exchange, Cl-/HCO3- exchange, and Cl-/oxalate exchange. To study its role in kidney and intestinal physiology, gene targeting was used to prepare mice lacking Slc26a6. Homozygous mutant Slc26a6-/- mice appeared healthy and exhibited a normal blood pressure, kidney function, and plasma electrolyte profile. In proximal tubules microperfused with a low-HCO3-/high-Cl- solution, the baseline rate of fluid absorption (Jv), an index of NaCl transport under these conditions, was the same in wild-type and null mice. However, the stimulation of Jv by oxalate observed in wild-type mice was completely abolished in Slc26a6-null mice (P<0.05). Formate stimulation of Jv was partially reduced in null mice, but the difference from the response in wild-type mice did not reach statistical significance. Apical membrane Cl-/base exchange activity, assayed with the pH-sensitive dye BCPCF in microperfused proximal tubules, was decreased by 58% in Slc26a6-/- animals (P<0.001 vs. wild types). In the duodenum, the baseline rate of HCO3- secretion measured in mucosal tissue mounted in Ussing chambers was decreased by approximately 30% (P<0.03), whereas the forskolin-stimulated component of HCO3- secretion was the same in wild-type and Slc26a6-/- mice. We conclude that Slc26a6 mediates oxalate-stimulated NaCl absorption, contributes to apical membrane Cl-/base exchange in the kidney proximal tubule, and also plays an important role in HCO3- secretion in the duodenum.

Animals↗

PubMed2004

Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine.

NHE3 Na(+)/H(+) exchanger knockout (Nhe3(-/-)) mice have severe absorptive deficits in the kidney proximal tubule and intestinal tract. The resulting hypovolemia has confounded efforts to carefully evaluate the specific effects of NHE3 deficiency on kidney function. Development of mice with transgenic expression of NHE3 in the small intestine (tgNhe3(-/-)) has allowed us to analyze the role of renal NHE3 in overall maintenance of blood pressure, pressure natriuresis, and autoregulation of both glomerular filtration rate (GFR) and renal blood flow (RBF). Ambulatory blood pressure, measured by telemetry, was lower in tgNhe3(-/-) mice than in wild-type controls (tgNhe3(+/+)) when the mice were maintained on a normal NaCl diet but was normalized when they were provided with a high NaCl intake. Furthermore, administration of the AT1-receptor blocker losartan showed that circulating ANG II plays a major role in maintaining blood pressure in tgNhe3(-/-) mice fed normal NaCl but not in those receiving high NaCl. Clearance studies revealed a blunted pressure-natriuresis response in tgNhe3(-/-) mice at lower blood pressures but a robust response at higher blood pressures. Autoregulation of GFR and RBF was normal in tgNhe3(-/-) mice. These results show that dietary NaCl loading normalizes blood pressure in awake tgNhe3(-/-) mice and that alterations in NHE3 activity are not essential for normal autoregulation of GFR and RBF. Furthermore, the data strongly support the hypothesis that NHE3 plays an important role in the diuretic and natriuretic responses to increases in blood pressure but also show that mechanisms not involving NHE3 mediate pressure natriuresis in the higher range of blood pressures studied.

Angiotensin II↗

PubMed2004

In vivo and in vitro analysis of cardiac troponin I phosphorylation.

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.

Animals↗

PubMed2004

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart.

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.

Animals↗

PubMed2004

The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro.

Although ouabain is known to induce hypertension, the mechanism of how this cardiac glycoside affects blood pressure is uncertain. The present study demonstrates that the alpha2-isoform of the Na-K-ATPase mediates the pressor effects of ouabain in mice. To accomplish this, we analyzed the effect of ouabain on blood pressure in wild-type mice, where the alpha2-isoform is sensitive to ouabain, and genetically engineered mice expressing a ouabain-insensitive alpha2-isoform of the Na-K-ATPase. Thus differences in the response to ouabain between these two genotypes can only be attributed to the alpha2-isoform of Na-K-ATPase. As the alpha1-isoform is naturally resistant to ouabain in rodents, it will not be inhibited by ouabain in either genotype. Whereas prolonged administration of ouabain increased levels of ouabain in serum from both wild-type and targeted animals, hypertension developed only in wild-type mice. In addition, bolus intravenous infusion of ouabain increased the systolic, mean arterial, and left ventricular blood pressure in only wild-type anesthetized mice. In vitro, ouabain increased vascular tone and thereby phenylephrine-induced contraction of the aorta in intact and endothelium-denuded wild-type mice but in alpha2-resistant mice. Ouabain also increased the magnitude of the spontaneous contractions of portal vein and the basal tone of the intact aorta from only wild-type mice. The increase in aortic basal tone was dependent on the presence of endothelium. Our studies also demonstrate that the alpha2-isoform of Na-K-ATPase mediates the ouabain-induced increase in vascular contractility. This could play a role in the development and maintenance of ouabain-induced hypertension.

Anesthesia↗