Sodium pump isozymes are differentially expressed in electrically dissimilar regions of colonic circular smooth muscle.

Abstract

Molecular analyses of NaK-ATPase abundance and a-subunit isoform distribution were performed to determine whether pump expression varies at different points through the thickness of the circular layer of colonic smooth muscle. The mRNA and polypeptides of Na,K-ATPase al and 13 subunits were twice as abundant in the submucosal region of the circular layer, which has previously been shown to generate large pump potentials. Sodium pump activity directly correlated with the relative abundance of the al polypeptide. These data show that sodium pump expression varies in electrically dissimilar regions of the circular layer.Recent studies have suggested that the gradient in resting potential across the circular muscle layer of the canine colon is due to a gradient in the contribution of the Na,K-ATPase to the resting potential (1). Drugs or ionic conditions that inhibit the Na,K-ATPase abolished the resting-membrane potential gradient, such that all cells were polarized to approximately -40 mV (1). Several differences in cells through the circular layer might be responsible for the gradient in the contribution of the sodium pump to membrane potential. There may be differences in (i) pump expression in submucosal vs. myenteric muscle cells, (ii) membrane conductance in the two regions, (iii) concentration of pump substrates such as intracellular Na', or (iv) Na' influx pathways such as differential expression of electrically silent transporters (e.g, Na+/H' exchange).The Na,K-ATPase couples the hydrolysis of ATP to the transport of three Na' ions out of the cell and two K+ ions into the cell for each pump cycle (2,3). This stoichiometry is the basis for its electrogenicity. Na,K-ATPase consists of a catalytically active a subunit (Mr = 112,000) and a glycosylated 13 subunit (protein component, Mr = 35,000). Several laboratories have identified cDNAs encoding three isoforms of the a subunit in rat brain [referred to as al, a2 (or a+), and a3] (4-7). Na,K-ATPase isozymes, which are composed of one a subunit (al, a2, or a3) and one , 1 subunit, have different affinities for sodium (8,9). Thus distribution of the isozymes in the circular muscle layer should be determined, because differential expression of these isoforms of the pump may affect the magnitude of pump current and, therefore, the contribution of the sodium pump to resting potential. Experiments in the present study demonstrate the following: (i) at the RNA level, Na,K-ATPase al isoform and 13 subunit mRNAs are at least twice as abundant in submucosal circular muscle as in myenteric circular muscle whereas the a2 isoform is equally distributed throughout the circular muscle layer, (ii) at the protein level, Na,K-ATPase al protein is more abundant in submucosal circular muscle than in myenteric circular muscle whereas the a2 isoform is equally distributed, and (iii) there is approximately twice as much sodium pump activity in submucosal circular muscle as in myenteric circular muscle. MATERIALS AND METHODSTissue Preparation. Mongrel dogs of either sex wer...

Different sensitivity of Na(+),K(+)-ATPase and Mg(2+)-АТРase to ethanol and arachidonic acid in rat colon smooth muscle under pretreatment of cellular membranes with Ds-Na

“…The minimal required conditions are the keeping of the optimal detergent/protein ratio, protection of the enzyme active center by substrate ATP and chelator presence in the case of pretreatment with negatively charged Ds-Na. It should be emphasized that CSM Na + ,K + -ATPase activity (up to 30 µmoles of Р i /hour per 1mg of protein) is appreciably higher that is known for other smooth muscles including myometrium [11,22]. This indicates the necessity for compliance with mild pretreatment procedure at room temperature, proper detergent/ protein ratio and considerable detergent dilution in ATPase medium far below CMC Summarizing in general, it is important to emphasize that the basic principles of the methodological procedure are determined by standard characteris tics and precaution of commonly used detergents in membranological studies [15,16,20,21] requiring mild pretreatment procedure at room temperature before incubation of permeabilized membranes with effectors or in ATPase medium at 37 °C.…”

“…The universal methodical approach, proven in other tissues [15][16][17], is adapted in our case. It provides simple and reliable detection of the CSM Na + ,K + -АТРase functional activity in a relatively nonpurified membranes -the highest of the known for smooth muscle enzyme, including myometrial plasma membranes [11,22], with comparable Mg 2+ -АТРase acivity. The technique is appropriate for membranological assays, quantitative comparison in pathophysiological studies and for testing the impact of diverse effectors on Na + ,K + -АТРase.…”

“…Na + ,K + -ATPase from different sources is used as an appropriate in vitro model for evaluation of the inhibitory potential and specificity of the membrane-acting biologically active compounds, applied in medical, pharmacological and environmental researches [9,10]. To elucidate the functional involvement into the pathophysiological mechanisms the Na + ,K + -ATPase expression is studied along with enzymatic detection in crude membrane preparation [4,11].…”

The influence of iron ions on ATP-hydrolases activity of cell membranes of rat colon smooth muscle and kidney

“…Следует подчеркнуть, что Na + ,K + -AТРаза по-чек представлена исключительно α1-изоформой [7,8], которая превалирует и в ГМОК, где, оче-видно, присутствует дополнительная минорная уабаинчувствительная изоформа [18,19], что характерно и для ряда других гладких мышц [30,31]. Однако влияние изоэнзимного соста-ва на специфику окислительной инактивации Na + ,K + -AТРазы в препаратах ГМОК и почек не является очевидным.…”

“…Функциональные нарушения Na + ,K + -AТРазы происходят при развитии ряда патологий по-чечного эпителия, в том числе при ишемии, со-провождаясь снижением ее активности [16,17]. Кроме того, Na + ,K + -AТРаза почек является го-могенной по изоформному составу каталитиче-ской субъединицы и содержит исключительно α1-изоформу [7,8], что важно при сравнении с энзимными комплексами с малоизученным изо-энзимным составом, в частности из гладких мышц ободочной кишки (ГМОК) [18,19], и воз-можной спецификой чувствительности изоформ к прооксидантам [20].…”