Role of Na + -K + ATPase enzyme in vascular response of goat ruminal artery

Indian Journal of Pharmacology, Mar/Apr 2009 by Kathirvel, K, Parija, S

Introduction

Na ,K -ATPase is an enzyme of the plasma membrane of most cells that uses cellular ATP to exchange cytoplasmic Na for extracellular K . [1] The function of the Na,K-ATPase is essential for the generation and maintenance of the electrochemical gradients. [2] Structurally, Na ,K -ATPase is an oligomer that is composed of distinct molecular forms of two major polypeptides, the α and β subunits. [3] At present, four structural variants of the α polypeptide (α1 , α2 , α3 and α4 ) and three b (b1 , b2 and b3 ) subunits have been identified in mammals. Association of the a and β polypeptides in different oligomers results in multiple isozymes of the Na ,K -ATPase that have unique functional properties and a tissue-specific pattern of expression. [3]

The sodium pump, in turn, is the target for multiple regulatory mechanisms. [4] It is also responsible for maintaining tone and contractility of smooth muscle. [5] Ouabain, a cardiotonic steroid, has been shown to be an endogenous factor that is secreted by the adrenal glands in humans and other mammals and is present in blood at nanomolar concentrations. [6] The mechanism of action of ouabain has been attributed classically to ion changes that are secondary to inhibition of the catalytic and transport activity of the Na ,K -ATPase. [7] Four different isoforms, namely α1 , α2 , α3 and α4 with different ouabain affinities are expressed in different species and in a tissue-specific manner. [8] Regional variations in activity and isoform-specific expression of sodium pump in vascular tissues have been reported. [9] The presence of a microsomal Na ,K -ATPase in sheep rumen epithelium activity which is reduced by 50% in the presence of ouabain has been reported. [10] Studies using biopsies of rumen epithelium papillae measured a net influx of [ 86 Rb] across the canine ruminal epithelium [11] and these findings are similar to a high concentration of Na , K -ATPase found in [ 3 H] ouabain-binding studies. [12] But there is no research related to ruminal artery to date on Na , K -ATPase and its role in maintaining the vascular tone in ruminal artery. Therefore, the present study was undertaken to identify the sensitivity of Na , K -ATPase to ouabain in goat ruminal artery.

Materials and Methods

Preparation of ruminal arterial ring and tension recording

After careful exposure of the goat celiac artery, the branch of the right ruminal artery was dissected out and placed in a cold aerated modified Krebs-Henseleit saline (MKHS) solution. Arteries were cleared of fat and connective tissue and cut into rings of about 2-2.5mm in length. The arterial ring was then mounted between two stainless steel L-shaped hooks made of 28 gauge stainless steel wire and kept under resting tension of 2g in a thermostatically controlled (37.0 ± 0.5 o C) automatic organ bath (Pan Lab) of 20ml capacity, containing MKHS and was aerated continuously with air. The arterial rings were equilibrated for 1.5h before recording the muscle tension. During this period, the bathing fluid was changed every 15 min. The change in tension was measured by a highly sensitive isometric force transducer (Model: MLT 0201, AD instruments, Australia) and recorded in a PC using chart 5.0 Pro software.

ACh (10 -6 M) on 5-HT- induced sustained contractile response

In order to examine intact functional endothelium, a single sub-threshold dose of 5-HT (1µM) was added to the bath after equilibration. ACh (10µM) was added to the bath with contact period of 1 min as soon as 5-HT induced contractile response was maintained at plateau. The effect of ACh (10µM) on the 5-HT (1µM) - induced sustained contractile response was examined considering the 5 HT- induced contraction at plateau as 100%. This procedure was followed in each experiment to study Na ,K -ATPase activity. The functional activity of Na ,K -ATPase was indirectly measured using the method described by Webb and Bhor. [13] In brief, in the absence of ACh effect on 5-HT induced sustained contraction, the bath solution was replaced by modified KHS and arterial ring was incubated for 30 min and solution changed at 15 min intervals for further experiments.

Experiments with K -free physiological solution

K -induced relaxation of arterial segments exposed to K -free MKHS is an experimental protocol for functional assessment of vascular sodium pump. In order to study the regulation of the ruminal artery sodium pump by vascular endothelium and different protein kinases, tissues were equilibrated in MKHS for 90 min and then the tissue viability was checked with 5-HT (1.0µM). The endothelial integrity was examined by applying ACh (10µM) to the vessels pre-constricted with 5-HT (1 µM). Then, after a period of wash for 30 min in MKHS, tissues were incubated in K-free solution and all the subsequent experimental protocols were carried out in K - free solution. Incubation of ruminal arterial segment in K -free solution generated a rise in basal tension which was not stable (and was noted in 2-3 experiments). In order to obtain a stable contraction in K - free medium submicromolar concentration of 5-HT was added to K - free PSS before the subsequent experimental protocol.