Isoform-specific Regulation Of Na/K Pump And Its Mechanism By Adrenergic Agonists In Rat Cardiocytes

The Na/K pump (NKP) is a ubiquitous plasma membrane enzyme, it belongs to the family of P-ATPase. The enzyme pumps 3Na+ and 2K+ ions against their concentration gradient, at the expense of an ATP molecule. In addition, it provides the driving force for the transport of other solutes, notably amino acids, sugar, and phosphate. The nonequivalent transport is electrogenic and leads to the generation of a transmembrane electrical potential allowing cells to become excitable. An altered enzyme activity occurs in a number of clinical disorders, such as hypertension, diabetes and other diseases. Therefore, elucidating the regulatory mechanisms that underlie the control of NKP activity has become an important issue.Adrenergic receptors (ARs) are known to regulate NKP activities. And the AR family was first divided into two subtypes, theα- andβ-ARs as determined by pharmacological studies in isolated tissue. Many studies have illustrated that the both two receptors can regulate NKP. The regulation of NKP in various tissues is under the control of a number of circulating hormones that impart both short- and/or long-term control over its activity, which molecular mechanisms are still unclear. Understanding the mechanisms underlying short- and long-term regulation of the NKP is essential for analyzing the adaptation of cells and tissues to the endocrine and electrolyte status of the organism, as well as the developing treatment for pathophysiological disorders caused by the functional abnormality of NKP. The present study aims to explore the effects of short- and long-term AR activation on the NKP activity and its mechanism.Objective: Na/K pump (NKP) can be regulated by many factors, and these regulations can divide into short-term and long-term regulation. Thus, we investigated the regulation of adrenoceptor activation on NKP and its mechanism in rat cardiocytes.Method: (1) Acute myocyte isolation: Sprague—Dawley rat cardiocytes were enzymatically isolated and kept in'Kraft-Brühe Solution'. Then cells were incubated with norepinephrine (NA) (propranolol was co-incubated with NA to blockβ-AR) and isoproterenol (ISO), respectively, for short-term (10min) and long-term (24h) at room temperature.(2) Determination of NKP current: the whole-cell patch-clamp technique were used to observe the effects ofα- andβ-AR agonists on NKP current (I_p).(3) Extraction of whole cell protein, plasma membrane protein and endosomes: the isolated myocytes were firstly incubated with drugs, then lysed with ice-cold lysis buffer to obtain the whole cell protein. We use the Cell Surface Protein Isolation Kit (Thermo) to extract the plasma membrane protein according to manufacturer's guidelines. Early endosomes (EE) and late endosomes (LE) were fractionated by a sucrose gradient method. The BCA protein assay kit was used to determine the concentration of proteins.(4) Western Blotting: equal amounts of proteins were separated by electrophoresis on a 10% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked 1h at 37°C in 5% nonfat dry milk in TBST. NKP anti-α₁ (Santa, 1:500) and NKP anti-α₂ (Upstate, 1:200) were used as primary antibodies. Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (KPL, 1:1000) were used as secondary antibodies. Blots were developed using the enhanced chemiluminescent method.(5) Extraction of mRNA: total RNA was extracted from rat cardiocytes using a Promega SV Total RNA Isolation System (Promega) according to manufacturer's guidelines.(6) Reverse transcription polymerase chain reaction (RT-PCR): the reverse transcription product (cDNA) was amplified by PCR using NKPα₁- andα₂-isoform specific primers. The PCR products were analyzed by agarose gel electrophoresis (2% agarose) followed by GoldViewâ…¡nucleic acid staining. (7) Relative Quantification of Specific cDNA by Real-time PCR: GAPDH was also chosed as the house-keeping gene. Real-time PCR was performed simultaneously for NKPα₁-isoform andα₂-isoform, GAPDH on cDNA. PCR products were analyzed on 2% agarose gel.Result: (1) Adrenoceptor short-term activation can affect I_p by an isoform-specific manner. The activation ofα-AR increased the high affinity pump current (I_h) by specifically increasing the activity of the high glycoside affinityα₂-isoform of NKP but did not change the low affinity current (I_l) induced byα₁-isoform; the activation ofβ-AR reduced the I_l by specifically decreasing the activity of the low glycoside affinity NKPα₁-isoform but did not influence the I_h induced byα₂-isoform.(2) Whateverα- orβ-AR activation, no significant changes in mRNA level and protein abundance of NKPα-isoform were observed in whole cell homogenates for AR short-term activation.(3) The short-term activation ofα-AR increased the plasma membrane protein abundance of NKPα₂-isoform, but did not affect theα₁-isoform protein abundance; the short-term activation ofβ-AR induced a reduction in the plasma membrane protein abundance of NKPα₁-isoform, but did not affect theα₂-isoform protein abundance.(4) The effect of short-term activation ofα- andβ-AR on NKP activity could be abolished by phalloidin which blocks the translocation of NKPα-isoform on plasma membrane.(5)α-AR short-term activation can regulate NKP activity through PKC pathway, andβ-AR short-term activation can regulate NKP activity through PKA pathway.(6) Adrenoceptor long-term activation can affect the density of I_p by an isoform-specific manner. The activation ofα-AR reduced the density of I_h by specifically decreasing the activity of the high glycoside affinityα₂-isoform of the NKP, but did not change I_l induced byα₁-isoform; the activation ofβ-AR increased the density of I_l by specifically increasing the activity of the low glycoside affinityα₁-isoform of the NKP, but did not change I_h induced by α₂-isoform.(7) The long-term activation ofα-AR reduced the protein expression of NKPα₂-isoform, but had no effect onα₁-isoform protein expression;β-AR long-term activation induced an increasing in the protein expression of NKPα₁-isoform, but had no effect onα₂-isoform protein expression.(8)α-AR long-term activation could reduce the level of NKPα₂-isoform mRNA, but had no effect onα₁-isoform mRNA level;β-AR long-term activation could induce an increasing in the level of NKPα₁-isoform mRNA, but had no effect onα₂-isoform mRNA level.(9)α-AR long-term activation regulated NKP activity through PKC pathway, andβ-AR long-term activation regulated NKP activity through PKA pathway.Conclusion: These results suggest the effects of short- or long-term adrenergic activation on NKP activity in heart isαisoform-specific. The activation ofα-AR affects I_h through a PKC-dependent pathway, which coupled with the high glycoside affinityα₂ isoform of NKP. In contrast, the activation ofβ-AR influences I_l through a PKA-dependent pathway, which coupled with the low glycoside affinityα₁ isoform of NKP. However, whateverα- orβ-adrenergic activation, the short-term actions on NKP induced a negative feedback on their long-term effects. The short-term regulation is mediated by the translocation of the NA/K pumpα-isoform; and the long-term regulation is mediated by the changes of protein expression and the levels of mRNA.