Pretreatment of human platelet membranes with trypsin abolishes GTP but not Na+ effects on alpha 2-adrenoreceptor-agonist interactions

The affinity of many types of membrane-bound receptors coupled negatively to adenylate cyclase is regulated by divalent and monovalent cations and by guanine nucleotides (GTP). We used alpha 2-adrenoreceptors of human platelets as a model system to find out the effect of limited proteolysis with trypsin on the regulation of the alpha 2-adrenoreceptor-agonist interactions by GTP and Na+. We found that partial proteolysis of the membranes with trypsin for 3 min at 35 degrees C reduced specific [3H]yohimbine binding to platelet membranes to 40-50% of control. The following characteristics of the receptors remaining after proteolysis were similar to those of untreated membranes: affinity for the agonist and antagonists, stereospecificity, and kinetic properties. Trypsin also did not modify the ability of the receptor's change from a high to low affinity state in the presence of Na+. These findings suggested that the capability of the receptors to recognize the ligand and their ability to undergo a conformational change in the presence of the agonist were retained despite a reduction in the total number of receptors by trypsin. However, the modulation of the receptor--agonist interactions by GTP or Mg2+ was lost in the trypsin-pretreated membranes, while the modulation by Na+ remained intact. It is suggested that the loss of GTP or Mg2+ effects on receptor--ligand interactions produced by trypsin may be due to trypsin-induced disruption of subunits (alpha i, beta gamma) interactions of Gi protein.     

Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding

The affinity of many types of membrane receptors for agonists is decreased by Na+ in radioligand binding experiments. We studied the alpha 2-adrenergic receptor of human platelets to determine whether Na+ acts at an intracellular or extracellular location. The Na+ content of intact platelets in an isotonic saline buffer was 38 nmol/10(8) platelets. This increased to 138 nmol/10(8) platelets with the Na+-selective ionophore monensin and decreased to 13 nmol/10(8) platelets with incubation in a Na+-free buffer. Epinephrine-induced platelet aggregation was increased by the addition of monensin and was decreased in the Na+-free buffer, while thrombin-induced aggregation was unaltered by either condition. Monensin, gramicidin, and ouabain (which all increased intraplatelet Na+) caused a 2-3-fold increase in the Kd of epinephrine (in competition with [3H]yohimbine) for alpha 2-adrenergic receptors on intact platelets. Conversely, incubation in a Na+-free buffer (which decreased intraplatelet Na+) decreased the Kd of the receptors for epinephrine 2-3-fold. These experiments suggest that changes in intracellular Na+ alter epinephrine binding. Control studies eliminated several alternative explanations for the effect of monensin on epinephrine binding: 1) monensin altered epinephrine binding only with intact platelets and not with platelet membranes; 2) although monensin depolarized platelets (assessed by [3H]methyltriphenylphosphonium uptake), other depolarizing conditions did not change epinephrine binding; 3) although monensin may increase intracellular pH (by exchanging Na+ for H+) such an increase in pH decreased the Kd of alpha 2-receptors on platelet membranes for epinephrine, an effect opposite to that produced by monensin in intact platelets. We conclude that alterations in the intracellular concentration of Na+ may change the affinity of platelet alpha 2-receptors for epinephrine. These results suggest a key role for intracellular Na+ in modulating binding at cell surface receptors in vivo.     

Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at alpha(1a)-adrenergic receptors

We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.     

Inhibition and derivatization of the renal Na,K-ATPase by dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.     

Epibatidine activates muscle acetylcholine receptors with unique site selectivity.

We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.     

Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.     

Rat brain and heart muscarinic receptors: modification with tetranitromethane

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.     

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.     

Rat ovarian angiotensin II receptors. Characterization and coupling to estrogen secretion

Angiotensin II receptor agonist (125I-angiotensin II) and antagonist (125I-[Sar1,Ile8]angiotensin II) bind in a specific and saturable manner to rat ovarian membranes. Agonist and antagonist binding affinity (KD approximately 0.5 nM) and the number of sites estimated (Bmax approximately 60 fmol/mg of protein) were similar. Dissociation of receptor-bound agonist was more rapid than the dissociation of receptor-bound antagonist, and agonist, but not antagonist, dissociation from the receptor was accelerated by GTP gamma S. A 0-150 mM increase in Na+ produced a 27% increase in the KD of agonist binding. Antagonist binding was not modified by Na+. These studies suggest that both agonist and antagonist identify putative angiotensin II receptors in the ovary but that the properties of agonist and antagonist binding are distinct. Angiotensin II antagonist binding sites are present on the granulosa cell layer of rat ovarian follicles (Speth, R. C., Bumpus, F. M., and Husain, A. (1986) Eur. J. Pharmacol. 130, 351-352). To determine the role of angiotensin II in ovarian function, we examined angiotensin II receptors and function during the onset of puberty. High affinity and low capacity angiotensin II receptors were present in ovaries from immature rats. After pregnant mare's serum gonadotropin induced ovulation in immature rats, antagonist binding to total ovarian membranes increased over 3-fold. In vitro incubation of peripubertal ovaries with 1 microM angiotensin II produced a stimulation of estrogen, but not progesterone, secretion. This steroidogenic effect of angiotensin II was most pronounced in the luteal phase of the estrus cycle. These studies point toward the involvement of angiotensin II in the regulation of ovarian function, possibly through modulation of follicular estrogen levels.     

Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides

Differences in binding properties of mu and delta opioid receptors were investigated using DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), which occur, respectively, as the most selective mu and delta radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two-site competitive model was adequate to explain the interactions of DAGO and DTLET with [3H]DTLET and [3H]DAGO binding sites, respectively. The weak cross-reactivity (congruent to 10%) of DTLET for mu sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35 degrees C, the lifetime of DTLET within its receptor site is about 14 times longer than that of the mu agonist. Sodium and manganese ions decrease the maximal number of high affinity mu and delta sites, but the sensitivity of mu receptors is three times higher towards Na+ and 20-fold higher towards Mn2+ than that of delta receptors. GTP reduces similarly the mu and delta binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP-P(NH)P]. However, in the presence of Na+ ions, GMP-P(NH)P inhibits the DTLET binding in a concentration-dependent manner. The effects of Na+ and GMP-P(NH)P could be explained by a sequential transformation of delta receptors to low-affinity states. This model predicts that Na+, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high-affinity delta receptors and that GMP-P(NH)P enhances the Na+ effect. Moreover, the binding kinetic to this high-affinity state was also modified by Na+ and nucleotides. All of these data support the existence of two independent mu and delta binding sites, the properties of which are differentially regulated by these endogenous effectors.     

Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+

The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities.     

Inositoltriphosphate receptors and ryanodine receptors in regulation of cholinosensitivity of Helix lucorum neurones by Na,K-pump during habituation

Influence of ouabain, the inhibitor of Na,K-pump, on habituation of Helix to tactile stimulation was identical to the ouabain-induced modification of cholinosensitivity reduction in command neurones of defensive behaviour of Helix lucorum in cellular model of habituation. Effects of intracellularly injected ligands of two types of Ca2+ -depot receptors, inositoltrisphosphate (IP3) and ryanodine receptors, on ouabain-induced changes were studied in cellular model of habituation. The antagonist of IP3 receptors heparin (0.1 mM), their agonist IP3 (0.1 mM) and inhibitor of ryanodine-dependent Ca2+ mobilization dantrolen (0.1 mM) prevented the depression of acetylcholine-induced current from the ouabain-evoked modification. The agonist/antagonist of ryanodine receptors ryanodine at two tested concentrations (0.1 mM and 1 mM) did not change the ouabain effect. It is concluded that Ca2+ released from intracellular Ca2+ -depots via IP3 receptors is involved into neuronal mechanism of Na,K-pump regulation of habituation in Helix lucorum to tactile stimulation.     

Na+/H+ exchangers, alpha-2-adrenergic receptors, sodium sensitivity and arterial hypertension]

Existing evidences indicate that a crossed regulation between alpha 2-adrenergic receptors and Na+/H+ exchanger(s) exists, that Na decreases the affinity of alpha 2-adrenergic receptors for agonists and antagonists, that intracellular Na+ and H+ ion concentrations regulate Na+/H+ exchanger activity, that intracellular pH controls the affinity of the alpha 2-adrenergic receptors for their agonists and antagonists. Alterations of alpha 2-adrenergic receptor densities and allosteric regulation by sodium have been demonstrated in sodium-dependent hypertension in rats. Increased Na+/H+ exchanger activity has been reported in genetic hypertension. Nevertheless, cosegregation experiments and human genetic polymorphism suggest that the exchanger could not be related to hypertension. We propose the following hypothesis: the increased Na+/H+ exchanger characteristic of hypertension could be secondary to the abnormalities of the alpha 2-adrenergic receptors found in hypertension, probably through the alteration of the sodium allosteric effect on these receptors.     

Essential role of magnesium in oxytocin-receptor affinity and ligand specificity.

We have analysed, with the aid of a novel radioiodinated oxytocin (OT)-receptor antagonist, the role of Mg2+ in uterine OT-receptor function. The antagonist-receptor interaction was characterized by high affinity, reversibility and stereospecificity in Tris/HCl buffer containing 3 mmol of Mg2+/litre as well as buffer free of Mg2+. By contrast, omission of Mg2+ decreased the affinity of the receptor for OT by about 1500-fold; moreover, the stereospecificity of agonist, but not antagonist, binding was lost. Since guanine nucleotides had relatively minor effects in this system (less than or equal to 2-fold decrease in OT affinity), we suggest that the agonist-binding site of OT receptors is directly modulated by Mg2+, unlike other receptors, where the effects of bivalent cations are exerted via guanine-nucleotide-binding (G-) proteins. Thus the ligand recognition mechanism of OT receptors may be novel in this respect.     

Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.     

Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.     

Alpha 2-adrenergic receptors and the Na+/H+ exchanger in the intestinal epithelial cell line, HT-29

alpha 2-Adrenergic receptors (alpha 2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to alpha 2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+ exchange elicited by alpha 2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of alpha 2-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+ uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 +/- 0.07 pH units/min/10(4) cells) was dependent on external sodium and was blocked by amiloride (Ki approximately 2.1 microM). Although epinephrine and the selective alpha 2-AR agonists clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective alpha 2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the alpha 2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they did not inhibit the amiloride-sensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as "amiloride-like" ligands selective for the Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, alpha 2-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the alpha 2-AR are tissue specific.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

Bradykinin modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule.

This paper studies the modulation by bradykinin of the ouabain-insensitive Na+-ATPase activity in both renal cortex homogenate and basolateral membrane from proximal tubule. The increase in bradykinin concentration from 10-14 to 10-10 M stimulated the ouabain-insensitive Na+-ATPase activity in cortex homogenates about 2.2-fold, but inhibited the enzyme activity of basolateral membrane preparations by 60%. In both preparations, the maximal effect was obtained with 10-10 M bradykinin. Further increase in the concentration of bradykinin completely abolished these effects. The antagonist of the B2 receptor, Hyp3, completely abolished the effect of 10-10 M bradykinin on the Na+-ATPase activity in the basolateral membrane preparation in a dose-dependent manner, but had no effect on the bradykinin stimulated enzyme activity of the cortex homogenate. Furthermore, in the presence of 10-7 M Hyp3, 10-10 M bradykinin stimulated the Na+-ATPase activity by 45% in the basolateral membrane preparations. The increase in des-Arg9-bradykinin concentration from 10-12 to 10-7 M, an agonist of the B1 receptor, stimulated the Na+-ATPase activity of the cortex homogenates and of the basolateral membrane preparations by 105 and 148%, respectively. In the presence of 25 microM mergetpa, an inhibitor of kininase I, the increase in bradykinin concentration from 10-12 to 10-10 M promoted similar inhibition of the Na+-ATPase activity of both cortex homogenates and basolateral membrane preparations. These results suggest that bradykinin stimulated the Na+-ATPase activity of proximal tubule through the interaction with B1 receptors and inhibited the enzyme through the interaction with B2 receptors. Furthermore, the cortex homogenate expresses a kininase I activity that cleaves bradykinin to des-Arg9-bradykinin.