Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes

Abstract

The role of phospholemman (PLM) in taurine and Cl(-) efflux elicited by 30% hyposmotic solution was studied in cultured cerebellar astrocytes with reduced PLM expression by antisense oligonucleotide (AO) treatment. PLM, a substrate for protein kinases (PK) C and A, is a protein that increases an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. Two antisense oligonucleotides (AO1 and AO2) effectively decreased the expression of the PLM protein by 40% and 30%, respectively, and markedly reduced [(3)H]taurine efflux by 67% and 62%. AO treatment also decreased the osmosensitive release of Cl(-), followed as (125)I. The inhibition of Cl(-) efflux (23% for AO1 and 13% for AO2) was notably lower than for [(3)H]taurine. The contribution of PKC and PKA in the function of PLM was also evaluated in astrocytes. Pharmacological activation or inhibition of PKC and PKA revealed that the osmosensitive taurine efflux is essentially PKC-independent while (125)I efflux is reduced by the PKC blockers H-7 (21%) and Gö6983 (41%). The PKA activator forskolin and dbcAMP increased taurine efflux by 66-70% and (125)I efflux by 21-45%. Norepinephrine increased the osmosensitive taurine efflux at about the same extent as dbcAMP and forskolin, and this was reduced by PKA blockers. These results suggest that PLM plays a role in RVD in astrocytes by predominantly influencing taurine fluxes, which are modulated by PKA but not PKC.

Effects of phospholemman downregulation on contractility and [Ca2+]itransients in adult rat cardiac myocytes

“…Using rather high concentrations (10 M) of AS oligonucleotides directed at the start codon or at nucleotides ϩ22 to ϩ46, Moran et al (12) demonstrated PLM downregulation in astrocytes by 40.5% and 27.2%, respectively, after 24 h of PLM AS oligonucleotide exposure. In addition, the regulatory volume decrease was markedly impaired in PLMdownregulated astrocytes (12), consistent with the ion channellike behavior (10), channel regulatory role (16), and osmotic regulatory function (11) ascribed to PLM in noncardiac tissues. In the present study, we did not use the AS oligonucleotide approach because it is well known that uptake of naked DNA by adult cardiac myocytes is a very inefficient process (8) and because we (19) have previously demonstrated that exposure to only 1 M of AS oligonucleotides (to NCX1) resulted in toxicity to adult rat cardiac myocytes.…”

“…To our knowledge, there has only been one published study (12) on the downregulation of PLM in neonatal rat cerebellar astrocytes. Using rather high concentrations (10 M) of AS oligonucleotides directed at the start codon or at nucleotides ϩ22 to ϩ46, Moran et al (12) demonstrated PLM downregulation in astrocytes by 40.5% and 27.2%, respectively, after 24 h of PLM AS oligonucleotide exposure.…”

“…We therefore reasoned that [Ca 2ϩ ] i transient and contractility changes associated with PLM downregulation would be sufficiently characterized with measurements at 0.6 and 5.0 mM [Ca 2ϩ ] o . Another limitation is that we ignored the observations in noncardiac tissues showing that PLM forms channels that are permeable to the zwitterion taurine (10) and participates in regulatory volume decrease of cells (11,12). Whole cell capacitance, however, was not changed in myocytes in which PLM was overexpressed (24) or downregulated (RESULTS).…”

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

“…Moreover, both when expressed in oocytes or added to lipid bilayers, PLM selectively transports the zwitterionic amino acid taurine (22), an osmolyte of animal cells. Recently, it was also shown that PLM stably transfected in HEK cells increases taurine efflux and regulatory volume decrease in response to cell swelling (39) and that in astrocytes taurine efflux can be inhibited after a decrease in the expression of endogenous PLM by antisense oligonucleotide (40). Thus, based on its function as a channel or a channel regulator, it is believed that PLM plays a specific role in muscle contractility and cell volume regulation.…”

Astrocytic Adrenoceptors: A Major Drug Target in Neurological and Psychiatric Disorders?

“…The astrocytic uptake of both GABA and taurine, another amino acid which is generally regarded as purely inhibitory, is stimulated by β-adrenergic receptor activation, but unaffected by α 1 or α 2 receptor agonists [42]. However, also the release of taurine is enhanced by activation of β-adrenergic receptors, both in bulk-prepared astrocytes [269] and in primary astrocyte cultures [270][271][272]. In the hypothalamo-hypohysial system the basal release of vasopressin and oxytocin from a perifused explant of the anterior lobe of the pituitary is enhanced by taurine [273].…”