Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.     

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.     

Inhibition of Na+/Ca2+ exchange in pituitary plasma membrane vesicles by analogues of amiloride

Amiloride is a weak inhibitor of Na+/Ca2+ exchange in isolated plasma membrane vesicles prepared from GH3 rat anterior pituitary cells. However, substitution on either a terminal guanidino nitrogen atom or the 5-amino nitrogen atom can increase inhibitory potency ca. 100-fold (I50 approximately 10 microM). A structure-activity study indicates that defined structural modifications of guanidino substituents are associated with increases in inhibitory activity. In contrast, analogues bearing 5-amino substituents generally increase in potency with increasing hydrophobicity of the substitution. Specificity in action of either class is indicated by several criteria. These inhibitors do not disrupt the osmotic integrity of the membrane, nor do they significantly interfere with plasmalemmal Ca2+-ATPase-driven Ca2+ uptake, Na+,K+-ATPase enzymatic activity, or the function of Ca2+ or K+ channels. Inhibition is freely reversible, further indicating a lack of nonspecific membrane effects. The mechanism by which each inhibitor class blocks exchange was found to be identical. Protonation of the guanidino moiety (i.e., cationic charge) is essential for activity. Analysis of transport inhibition as a function of Ca2+ concentration indicates noncompetitive kinetics. However, inhibition was reversed by elevating intravesicular Na+, indicating a competitive interaction with this ion. These results suggest that the inhibitors function as Na+ analogues, interact at a Na+ binding site on the carrier (presumably the site at which the third Na+ binds), and reversibly tie up the transporter in an inactive complex. In addition to blocking pituitary exchange, these analogues are effective inhibitors of the bovine brain and porcine cardiac transport systems.     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.     

Effects of amiloride analogues on Na+ transport in toad bladder membrane vesicles. Evidence for two electrogenic transporters with different affinities toward pyrazinecarboxamides

Most of the electrical potential-driven 22Na+ uptake in toad bladder membrane vesicles can be blocked by the diuretic amiloride. Analysis of the amiloride inhibition curve indicates the presence of two pathways with low and high affinities to the diuretic (Garty, H. (1984) J. Membr. Biol. 82, 269-279). The selectivity of these pathways to amiloride was explored by comparing the inhibition curve of this diuretic with those of 10 of its structural analogues. The relative potencies of various amiloride-like compounds as blockers of the flux component with high affinity to amiloride were in good agreement with the structure-activity relationships elucidated from transepithelial short-circuit current measurements. Thus, this pathway is most probably the apical Na+-specific channel. The other pathway with lower affinity to the diuretic was relatively insensitive to modifications of the amiloride molecule, and the structure-activity relationships measured for the inhibition of this pathway were different from those reported for any other amiloride-blockable process. Other experiments have established that the Na+ flux with low affinity to amiloride is electrogenic and is not mediated by a Na+/H+ or Na+/Ca2+ exchanger, Na+-hexose cotransporter, or the Na+/K+-ATPase. The data indicate that tracer flux measurements in toad bladder membrane vesicles monitor, in addition to the well-characterized apical Na+ channels, another amiloride-blockable electrogenic Na+ transporter. This pathway could be responsible for the basolateral amiloride-blockable Na+ conductance recently observed in nystatin-treated bladders (Garty, H., Warncke, J., and Lindemann, B. (1987) J. Membr. Biol. 95, 91-103).     

Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells

Addition of growth factors to responsive cells in HCO3- -free media results in a rapid rise in cytoplasmic pH (pHi) caused by activation of Na+/H+ exchange. In this paper, we have examined how pHi regulation and growth factor responsiveness are affected by HCO3(-)using quiescent mouse MES-1 fibroblastic cells as a model. When cells are exposed to 25 mM HCO3-, 5% CO2, steady-state pHi reaches a new more alkaline level (by 0.25 unit) within 10 min. This rise in pHi is both Na+- and HCO3- -dependent, does not occur in Cl(-)-depleted cells, and is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by 5-(n,n-dimethyl)-amiloride, indicating the involvement of Na+-dependent HCO3-/Cl- exchange. Furthermore, the recovery of pHi from acute acid loads is accelerated by HCO3- in a Na+-dependent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive manner and is blocked in Cl(-) -depleted cells. Similar results were obtained for mouse 3T3 cells and human fibroblasts. In the presence of HCO3-/CO2 (pH 7.35), mitogens and phorbol esters fail to induce a detectable rise in pHi. However, when steady-state pHi is artificially lowered by approximately 0.4 unit, growth factors evoke significant increases in pHi due to activation of Na+/H+ exchange. In the absence of HCO3-, mitogen-induced alkalinizations are readily detectable but not when pHi is artificially elevated to the value normally observed in HCO3- media. From these results we conclude that: 1) Na+-dependent HCO3-/Cl- exchange determines steady-state pHi and acts in parallel with Na+/H+ exchange to stimulate pHi recovery from acid loading; 2) Na+-dependent HCO3-/Cl- exchange raises steady-state pHi to a level beyond the operating range of the Na+/H+ exchanger and thereby prevents growth factors from alkalinizing the cytoplasm any further. The results also imply that, unlike Na+/H+ exchange, Na+-dependent HCO3-/Cl- exchange is not activated by mitogens.