Effects of streptozotocin-induced diabetes on leg muscle contractile properties and motor neuron morphology in rats.

Skeletal muscle fiber subtypes are differentially sensitive to diabetes-related pathology; For example, fast-twitch muscles exhibit severe decreases in contraction force while slow-twitch muscles demonstrate prolonged half-relaxation time. However, such alterations have only been examined after a relatively short period following diabetes onset, with no information available regarding muscle damage caused by longer disease periods (>20 weeks). This study examined alterations in the contractile properties of the medial gastrocnemius (fast-twitch) and soleus (slow-twitch) muscles, as well as morphological changes in their motor neurons 12 and 22 weeks after diabetes onset. Adult male Wistar rats were divided into diabetic (12- or 22-week post-streptozotocin injection) and age-matched control groups. Electrically evoked maximum twitch and tetanic tension were recorded from leg muscles. Additionally, motor neuron number and cell body size were examined. At 12 weeks after diabetes onset, decreases in twitch force were observed predominantly in medial gastrocnemius muscles, while soleus muscles exhibited prolonged half-relaxation time. However, these differences became ambiguous at 22 weeks, with decreased twitch force and prolonged half-relaxation time observed in both muscles. On the other hand, reduction in soleus motor neurons was observed 12 weeks after diabetes onset, while medial gastrocnemius motor neurons were diminished at 22 weeks. These data indicate that experimental diabetes induces differential damage to medial gastrocnemius and soleus muscles as well as motor neurons. These diabetes-induced differences may partly underlie the differential deficits observed in gastrocnemius and soleus.

Tissue-specific role of the Na,K-ATPase α2 isozyme in skeletal muscle.

The Na,K-ATPase α2 isozyme is the major Na,K-ATPase of mammalian skeletal muscle. This distribution is unique compared with most other cells, which express mainly the Na,K-ATPase α1 isoform, but its functional significance is not known. We developed a gene-targeted mouse (skα2(-/-)) in which the α2 gene (Atp1a2) is knocked out in the skeletal muscles, and examined the consequences for exercise performance, membrane potentials, contractility, and muscle fatigue. Targeted knockout was confirmed by genotyping, Western blot, and immunohistochemistry. Skeletal muscle cells of skα2(-/-) mice completely lack α2 protein and have no α2 in the transverse tubules, where its expression is normally enhanced. The α1 isoform, which is normally enhanced on the outer sarcolemma, is up-regulated 2.5-fold without change in subcellular targeting. skα2(-/-) mice are apparently normal under basal conditions but show significantly reduced exercise capacity when challenged to run. Their skeletal muscles produce less force, are unable to increase force to match demand, and show significantly increased susceptibility to fatigue. The impairments affect both fast and slow muscle types. The subcellular targeting of α2 to the transverse tubules is important for this role. Increasing Na,K-ATPase α1 content cannot fully compensate for the loss of α2. The increased fatigability of skα2(-/-) muscles is reproduced in control extensor digitorum longus muscles by selectively inhibiting α2 enzyme activity with ouabain. These results demonstrate that the Na,K-ATPase α2 isoform performs an acute, isoform-specific role in skeletal muscle. Its activity is regulated by muscle use and enables working muscles to maintain contraction and resist fatigue.

DOCA-salt hypertension does not require the ouabain-sensitive binding site of the α2 Na,K-ATPase.

BACKGROUND: We have shown that the ouabain-sensitive α2 Na,K-ATPase is required for adrenocorticotropic hormone (ACTH)-induced hypertension and gestational blood pressure regulation. It is therefore of interest to explore whether this binding site participates in the development of other forms of hypertension, such as deoxycorticosterone acetate (DOCA)-salt using mutant mice with altered sensitivity to ouabain. METHODS: Wild-type (α1 ouabain-resistant, α2 ouabain-sensitive: α(R/R)α2(S/S)), α1-resistant, α2-resistant (α1(R/R)α2(R/R)) and α1-sensitive, α2-resistant (α1(S/S)α2(R/R)) mice were uninephrectomized and implanted with DOCA pellets. The animals were given either tap water or 1% NaCl, and blood pressure was measured before and after DOCA. RESULTS: DOCA-salt-treated α1(R/R)α2(R/R) mice developed hypertension to the same extent as α1(R/R)α2(S/S) mice (wild type), and the α1(S/S)α2(R/R) mice given DOCA-salt also showed no difference from the other two genotypes. The expression of the α1 isoform was not changed by DOCA-salt treatment in either α1(R/R)α2(S/S) or α1(R/R)α2(R/R) mice. However, the α2 subunit was expressed at substantially higher levels in the hearts of α1(R/R)α2(R/R) than α1(R/R)α2(S/S) mice, regardless of treatment. Plasma levels of ouabain did not change consistently, but those of marinobufagenin were modestly higher in DOCA-salt treated mice relatively to those without salt. CONCLUSIONS: The ouabain-binding site of either the α1 or α2 Na,K-ATPase subunit does not play an essential role in the development of DOCA-salt hypertension in this mouse model. These findings indicate that the underlying mechanisms of hypertension induced by DOCA-salt treatment are different from those of ACTH-induced hypertension.

The ouabain-binding site of the α2 isoform of Na,K-ATPase plays a role in blood pressure regulation during pregnancy.

BACKGROUND: The cardiotonic steroid/ouabain-binding site of the α subunit of Na,K-ATPase is thought to play an important role in cardiovascular homeostasis. Previously, we demonstrated the cardiotonic steroid-binding site of the α2 Na,K-ATPase is involved in adrenocorticotropic hormone (ACTH)-induced hypertension by using gene-modified α2(R/R) mice in which the cardiotonic steroid-binding site is relatively resistant to ouabain compared to the ouabain-sensitive wild-type α2(S/S) mice. To further explore the importance of this site in the cardiovascular system, we investigated blood pressure regulation during pregnancy in mice with the α2(R/R) isoform. METHODS: The systolic blood pressure (SBP) of the α2(S/S) and α2(R/R) mice was measured before and during pregnancy by tail-cuff. The expression of the α isoforms of Na, K-ATPase in various tissues and plasma endogenous ouabain contents were assessed prior to pregnancy as well as days 7 and 17 of gestation. RESULTS: The α2(S/S) mice showed a gradual decrease in the SBP during the first two trimesters, followed by an increase above the preconceptional level in the third trimester. However, the α2(R/R) mice exhibited a lower blood pressure in the third trimester. The cardiac expression of the α2 Na,K-ATPase in the α2(S/S) mice was significantly less than that of the α2(R/R) mice throughout the pregnancy. The plasma endogenous ouabain concentration significantly increased by twofold at day 17 of pregnancy in the α2(R/R) mice but not in the α2(S/S) mice. CONCLUSIONS: The cardiotonic steroid-binding site of the α2 Na,K-ATPase plays a role in maintaining normal SBP during pregnancy.

Ala-504 is a determinant of substrate binding affinity in the mouse Na(+)/dicarboxylate cotransporter.

The Na(+)/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K(0.5) for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K(0.5) for both adipate and succinate.

Transmembrane helices 3 and 4 are involved in substrate recognition by the Na+/dicarboxylate cotransporter, NaDC1.

The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents. To identify domains involved in glutarate transport, we constructed a series of chimeric transporters between mouse and rabbit NaDC1. Although residues found in multiple transmembrane helices (TM) participate in glutarate transport, the most important contribution is made by TM 3 and 4 and the associated loops. The R(M3-4) chimera, consisting of rbNaDC1 with substitution of TM 3-4 from mNaDC1, had a decreased K(0.5)(glutarate) of 4 mM compared with 15 mM in wild-type rbNaDC1 without any effect on K(0.5)(succinate). The chimeras were also characterized using dual-label competitive uptakes with (14)C-glutarate and (3)H-succinate to calculate the transport specificity ratio (TSR), a measure of relative catalytic efficiency with the two substrates. The TSR analysis provides evidence for functional coupling in the transition state between TM 3 and 4. We conclude that TM 3 and 4 contain amino acid residues that are important determinants of substrate specificity and catalytic efficiency in NaDC1.

Functional characterization of high-affinity Na(+)/dicarboxylate cotransporter found in Xenopus laevis kidney and heart.

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na(+)/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is approximately 70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a K(m) for succinate of 4 muM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.