Labeling Of Phosphatidylinositol Research Articles

Gonadotropin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.

Steroidogenic effect of exogenous phospholipase C on bovine adrenal fasciculata cells

Phospholipase C (Bacillus cereus) added to the incubation medium stimulated the steroidogenic activity of bovine adrenal zona fasciculata cell suspensions to a level similar to that induced by optimal concentration of ACTH. This effect was not related to an increase of cyclic AMP; it was calcium-dependent and was also induced by an other bacterial phospholipase C (from Clostridium perfringens) whereas phospholipases A2 and D were ineffective. Phospholipid metabolism was examined in these cells after radiolabeling with [ 14C]-glycerol or [ 32P]orthophosphate. Phospholipase C induced a very fast (5 seconds) increase in cellular [ 14C]-1,2-diacylglycerol followed by [ 32P] labeling of phosphatidic acid and phosphatidylinositol. These events preceded the stimulation of steroidogenesis which was detectable after 2 minutes of incubation. These observations suggest that activation of an endogenous phospholipase C activity may be considered as an early event in the response of bovine adrenocortical cells to steroidogenic effectors such as angiotensin II and acetylcholine.

Phytate metabolism in Petunia pollen

Phytic acid has been detected in the anthers of young flower buds of Petunia hybrida, the amount increasing slowly as the flower develops until anther dehydration, when there was a more rapid increase in phytic acid content. In mature pollen, the phytic acid content was found to be 2.0 % by weight, of which 90 % was water soluble, while free myo-inositol was a relatively low 0.06 % by weight. Breakdown of phytic acid was initiated soon after pollen germination began, and its degradation products, myo-inositol and inorganic phosphate, were rapidly mobilized for phospholipid and pectin biosynthesis. Both are in high demand during pollen tube elongation. Utilization of myo-[2- 3H]inositol for phospholipid biosynthesis was about five times that for pectin synthesis during the first few hours of pollen germination. The label in the phospholipid was identified as the myo-inositol moiety of phosphaltidylinositol, while the pectin material contained predominantly labelled arabinose, with smaller amounts of label in galacturonic acid, glucose and xylose. A chase experiment showed that the myo-inositol moiety of phosphatidylinositol was subject to a relatively rapid turnover, while the label in pectin was not. Labelling germinating pollen with [ 32P]orthophosphate gave label in phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine of the phospholipids. Phosphatidylinositol contained 30 % of this label initially, a proportion which declined to 10 % over longer periods of germination.

The role of calcium in phospholipid turnover following glucose stimulation in neonatal rat cultured islets.

Phospholipid turnover was studied in cultured neonatal rat pancreatic islets. In islets prelabeled with [32P]Pi, 15-min stimulation with glucose (16.7 mM) caused increased labeling of phosphatidic acid (93%) and phosphatidylinositol (94%) and decreased labeling of the polyphosphoinositides (20%). Omission of calcium ion during the period of glucose stimulation did not modify the changes in inositol phospholipids. In islets equilibrated with [32P]Pi in the presence and absence of stimulatory glucose concentrations (11.1 and 1.7 mM, respectively), chelation of calcium by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increase in phosphatidic acid and phosphatidylinositol labeling. However, the decrease in polyphosphoinositide labeling was inhibited by the chelator only in islets labeled in the absence of stimulatory glucose concentrations, the decrease persisting in islets labeled in the presence of glucose. This suggests that a specific pool of polyphosphoinositides is labeled in the presence of agonist and decreases in response to acute glucose stimulation irrespective of availability of external calcium. In the absence of calcium, the addition of [gamma-32Pi]ATP to a membrane preparation of cultured islets yielded three lipid phosphorylation products (phosphatidic acid, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate). In broken cell preparations, [32P]Pi-labeled phosphatidylinositol was also detected. The extent of all these phosphorylations was decreased by the presence of free calcium ion (40 microM). These data indicate that polyphosphoinositide turnover takes place after glucose stimulation independent of extracellular calcium and support the possibility that this may play a primary role in altering cell calcium availability.

LHRH rapidly stimulates phosphatidylinositol metabolism in enriched gonadotrophs

The effects of luteinizing hormone-releasing hormone (LHRH) and human pancreatic growth hormonereleasing factor (hpGRF(l-40)-NH 2) on phospholipid metabolism were studied in rat anterior pituitary cells in primary culture. In a 4-fold enriched population of gonadotrophs, 30 nM LHRH increased 32P; incorporation into phosphatidic acid (PA) as early as l min after its addition. Phosphatidylinositol (PI) labeling was increased l min later. The stimulatory action of LHRH was observed in both phospholipids up to 100 min, the last time interval studied. The decapeptide did not affect 32P i labeling of phosphatidylcholine (PC), lysoPC, phosphatidylethanolamine or phosphatidylserine. Dose-response studies performed after 25 min of incubation showed an ED 50 value of LHRH action at approximately 1 nM for PI labeling. In contrast, the addition of 0.1 μM GRF to anterior pituitary cells enhanced 32P i incorporation only into PC after a 60 min incubation period. The present data suggest that stimulation of acidic phospholipid metabolism, particularly an increase in PA-PI turnover, may represent an early event in the mechanism of action of LHRH but not GRF in the anterior pituitary gland.

Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig cell steroidogenesis. II. Gonadotropin-releasing hormone stimulates phospholipid labeling.

To investigate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated Leydig cell steroidogenesis, the effects of GnRH agonist [des-Gly10, (D-Ala6) GnRH] on phospholipid turnover were studied. GnRH agonist in concentrations of 10(-9) to 10(-7)M increased phosphatidic acid labeling 292 +/- 16% (mean +/- SE), and phosphatidylinositol labeling 258 +/- 13.2%. GnRH agonist-stimulated phospholipid labeling was detectable as early as 2 minutes. GnRH antagonist completely blocked GnRH agonist-induced testosterone formation and phosphatidic acid and phosphatidylinosital labeling. Nifedipine in concentrations of 1 and 10 micrograms/ml inhibited GnRH agonist-stimulated testosterone formation but had no effect on 32P incorporation into phospholipids. Our results suggest that GnRH agonist-stimulated Leydig cell steroidogenesis is calcium dependent and correlated with increased phospholipid turnover.

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32P i into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10 −8 m GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 μ m PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 m m Ca 2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 m m Ca 2+ than at 1.2 m m Ca 2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca 2+ concentrations up to 1.5 m m. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca 2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.

Regulation of phosphatidylinositol turnover, calcium metabolism and enzyme secretion by phorbol dibutyrate in neutrophils

The action of the tumor promoter, phorbol 12,13-dibutyrate (PDBu), on rabbit peritoneal and human neutrophils is associated with stimulation of 14C-arachidonic acid incorporation into phospholipids within 1-2 min. Stimulated 14C-arachidonate incorporation was relatively selective for phosphatidylinositol (PI) in rabbit neutrophils. In contrast, the secretory response of human neutrophils to PDBu coincided with stimulated label incorporation into phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and PI. Significant increases in label incorporation were observed with PDBu concentrations as low as 2 nM, and the dose response of stimulated label incorporation paralleled that of evoked lysozyme secretion. A parallel, but partial, inhibition of PDBu-stimulated PI labeling and enzyme release was observed after exposing rabbit neutrophils to calcium-deprived medium, whereas calcium deprivation failed to significantly depress either of these stimulant actions of PDBu in human neutrophils. Further, in rabbit neutrophils PDBu elicited an increase in cell associated 45Ca. However, PDBu was unable to promote the incorporation of 32P orthophosphate into PI or enhance phospholipase A2 activity in broken cells. These findings suggest that one expression of the interaction between phorbol esters and their receptors on neutrophils involves the turnover of arachidonic acid in phospholipids. This stimulated turnover of arachidonate may be a critical step in the cascade of events associated with neutrophil activation.

Induction by lysophospholipids of CoA-dependent arachidonyl transfer between phospholipids in rat platelet homogenates

Rat platelet homogenates are able to catalyze CoA-mediated, ATP-independent transfer of arachidonic acid from platelet phospholipids to added lysophospholipids. Homogenates of platelets prelabelled with radioactive arachidonic or oleic acid were incubated in the presence of CoA and various lysophospholipids. Transfer observed with arachidonic acid-labelled platelets was dependent on the lysophospholipid added. When 1-alkenyl- or 1-acyllysophosphatidylethanolamine was used, there was a more efficient arachidonyl transfer from phosphatidylcholine than from phosphatidylinositol to the phosphatidylethanolamine fraction. Lysophosphatidylserine also accepted arachidonyl from phosphatidylcholine. Addition of lysophosphatidylcholine resulted in a decrease in the labelling of phosphatidylinositol and to a lesser extent of phosphatidylethanolamine with concomitant transfer to phosphatidylcholine. Lysophosphatidylinositol and lysophosphatic acid did not act as substrate for this transfer reaction. Free, non-radioactive arachidonic acid did not compete for the labelled arachidonic acid transfer. This pathway may play a major role in the synthesis of arachidonyl species of phosphatidylethanolamine and phosphatidylserine and for the arachidonyl transfer to the phosphatidylethanolamine plasmologen in stimulated platelets.

Modulation by thyroid status of cyclic AMP-dependent and Ca 2+-dependent mechanisms of hormone action in rat liver cells : Possible involvement of two different transduction mechanisms in α1-adrenergic action

The actions of hormones which are associated to cAMP-dependent and calcium-dependent mechanisms of signal transduction were studied in hepatocytes obtained from rats with different thyroid states. In cells from euthyroid and hyperthyroid rats, the metabolic actions of epinephrine were mediated mainly through α 1-adrenoceptors; beta-adrenoceptors seem to be functionally unimportant. In contrast, both α 1- and β-adrenoceptors mediate the actions of epinephrine in hepatocytes from hypothyroid animals. Phosphatidylinositol labeling was strongly stimulated by epinephrine, vasopressin and angiotensin II in cells from eu-, hyper- or hypothyroid rats. However, metabolic responsiveness to vasopressin and angiotensin II was markedly impaired in the hypothyroid state. The glycogenolytic response to the calcium ionophore A-23187 was also impaired, suggesting that hepatocytes from hypothyroid rats are less sensitive to calcium signalling. The persistence of α 1-adrenergic responsiveness in the hypothyroid state suggests that the mechanism of signal transduction for α 1-adrenergic amines is not identical to that of the vasopressor peptides. α 1-Adrenergic stimulation of cyclic AMP accumulation was not detected in cells from hypothyroid rats. These data suggest that factors besides calcium and besides cAMP are probably involved in α 1-adrenergic actions. Metabolic responses to glucagon and to the cAMP analogue dibutyryl cAMP were not markedly changed during hypothyroidism, although cAMP accumulation produced by glucagon and β-adrenergic agonists was enhanced. In hyperthyroidism, cell responsiveness to epinephrine, vasopressin, angiotensin II and glucagon was decreased, but sensitivity to cAMP was not markedly altered. The factors involved in this hyposensitivity to hormones during hyperthyroidism are unclear.

Rapid effects of angiotensin-II on polyphosphoinositide metabolism in the rat adrenal glomerulosa.

Angiotensin-II (A-II) provoked a rapid decrease in 32p in triphosphoinositide (TPI) in 32p-prelabeled rat adrenal glomerulosa cells. This effect (presumably reflecting TPI hydrolysis) of A-II was nearly maximal at 5 sec of incubation and appeared to precede increases in labeling of phosphatidic acid and phosphatidylinositol. Other aldosterone-stimulating agents (ACTH, K+ and serotonin) did not provoke this effect. Since this effect appeared to be independent of Ca++, it is possible that TPI hydrolysis may be important for Ca++ mobilization during A-II action in glomerulosa tissue.

Mode of cytotoxic action of pseudomonal leukocidin on phosphatidylinositol metabolism and activation of lysosomal enzyme in rabbit leukocytes.

The cytotoxic action of leukocidin from Pseudomonas aeruginosa was supported by the following observations. (i) The destruction of rabbit leukocytes by the toxin was reduced in the absence of Ca2+ and stimulated by the addition of calcium ionophore A23187 but inhibited by EDTA, EGTA, and TMB-8, an antagonist of intracellular Ca2+ transport. (ii) Uptake of 45Ca into leukocytes exposed to the toxin was enhanced about threefold the rate of uptake into untreated cells. The increased 45Ca uptake into the cells was slightly inhibited by trifluoperazine, an inhibitor of Ca2+-calmodulin activity, but not by ruthenium red. (iii) Pseudomonal leukocidin enhanced rapidly the labeling of phosphatidylinositol, polyphosphoinositides, phosphatidic acid, and lysophosphatidic acid from [32P]phosphate. The time course experiments of the labeling and breakdown of these phospholipids suggested that the initial action of this toxin was to stimulate phosphatidic acid production, presumably causing a rapid metabolic change of phosphatidylinositol correlating with the activities of phosphatidylinositol-specific phospholipase C and 1,2-diacylglycerol kinase. It was considered that a rapid formation of phosphatidic acid and degradation of polyphosphoinositides might be related to a Ca2+ movement from extra- and intracellular space. (iv) In leukocytes exposed to the toxin, acid phosphatase activity as a marker enzyme of lysosome was activated up to 75% of the lysosomal enzyme before cell destruction. The leakage of lysosomal enzyme from the cells occurred at the almost same time as leukocyte destruction. The mode of cytotoxic action of pseudomonal leukocidin is discussed.

Effect of insulin on alpha 1-adrenergic actions in hepatocytes from euthyroid and hypothyroid rats : Possible involvement of two pathways in alpha 1-adrenergic actions

The effect of insulin on the alpha 1-adrenergic stimulation of glycogenolysis and ureogenesis, which is very small or undetectable in hepatocytes from control animals, is marked in hepatocytes from hypothyroid rats; the metabolic actions due to alpha 1-adrenergic activation, but not those due to glucagon were nearly blocked by insulin in cells from hypothyroid rats. The alpha 1-adrenergic-mediated stimulation of phosphatidylinositol labelling was not affected by insulin in cells from either control or hypothyroid rats. The data suggest that the alpha 1-adrenergic action proceeds through two pathways, one of which is very sensitive to insulin and predominates in cells from hypothyroid rats.

Human pancreatic GRF stimulates phosphatidylinositol labeling in cultured anterior pituitary cells.

The effects of natural and synthetic human pancreatic growth hormone-releasing factor (hpGRF) on 32P incorporation into phospholipids were studied in cultured rat anterior pituitary cells. Natural hpGRF (1:30 dilution) significantly (P less than 0.01) stimulated phosphatidylinositol labeling at all times studied (15, 30, and 60 min). Synthetic hpGRF-(1-40)OH also significantly (P less than 0.05; P less than 0.01) increased 32P incorporation into phosphatidylinositol in a dose-related (1-30 nM) and time-dependent (5, 10, and 30 min) manner. In contrast, phosphatidylcholine and phosphatidylethanolamine labeling was not affected at any time studied. Somatostatin (30 nM) did not affect basal or hpGRF-stimulated phosphatidylinositol labeling but inhibited the hpGRF stimulation of cyclic AMP accumulation and growth hormone release. These results suggest that the phosphatidylinositol cycle may be involved in the mechanism of action of hpGRF in the anterior pituitary gland.

Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action.

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.

Stimulation by prostaglandin F 2α of phosphatidic acid-phosphatidylinositol turnover in rat luteal cells

Prostaglandin F 2α (PGF 2α) causes a rapid and marked increase of [ 32P]-orthophosphate incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min after PGF 2α addition into PA and PI, respectively, and by 10 min has reached a 2-fold stimulation over control in both lipid moieties. The labeling of other phospholipids is not affected. PGF 2α exerts its stimulatory effect at an ED 50 value of approximately 200 and 60 nM on PI and PA labeling, respectively. By contrast, human chorionic gonadotropin has no effect alone and does not interfere with the PGF 2α-induced stimulation of PA-PI labeling. The striking similarity between the effects of PGF 2α and LHRH on PA-PI labeling suggests that the two agents may exert their direct action on the corpus luteum via a common intracellular mechanism involving acidic phospholipid metabolism.

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca 2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N 6, O 2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca 2+ fluxes and increased 32PO 4 incorporation into phosphatidyl-inositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO 4 (the “pi effect”) was blocked when cell were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N, N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.

Sensitivity of liver cells formed after partial hepatectomy to glucagon, vasopressin and angiotensin II

During the active proliferation which follows partial hepatectomy, the sensitivity of liver cells to glucagon is markedly diminished. In hepatocytes obtained from rats partially hepatectomized 3 days before experiments were performed, the dose-response curves to glucagon were shifted to the right by about two orders of magnitude as compared to those of the control cells. Later on (7 days after surgery) the dose-response to glucagon was still shifted to the right but by only one order of magnitude. These data are consistent with the diminution in the number of glucagon receptors in liver plasma membrane during liver regeneration reported by other authors. No stimulation of glycogenolysis, gluconeogenesis or ureogenesis was produced by vasopressin or angiotensin II in hepatocytes from rats partially hepatectomized 3 days before experimentation. However, phosphatidylinositol labeling was stimulated in these cells to a similar extent as in the controls. The ionophore A23187 was also ineffective in stimulating glycogenolysis in these cells. Later, 7 days after surgery, the hepatic responsiveness to vasopressin and angiotensin II was restored. The data suggest that, during the initial stages of liver regeneration, the enzymatic machinery of the hepatocyte is not sensitive to calcium-signalling.

H 1-histaminergic activation stimulates phosphatidylinositol labeling in rabbit aorta

Histamine and 2-(2-aminoethyl)-thiazole induced dose-dependent increases in the labeling of phosphatidylinositol in rabbit aorta whereas impromidine was without effect. The effect of histamine was inhibited by antihistamines with the following order of potency: mepyramine ⩾ pyrilamine ⪢ cimetidine. The data indicate that H 1-histaminergic activation stimulates phosphatidylinositol labeling in rabbit aorta and suggest the involvement of a calcium-signaling process in the action of H 1-histaminergic receptors.

Muscarinic agonist binding and phospholipid turnover in brain.

The ability of muscarinic cholinergic agonists to interact with muscarinic receptors in nerve ending preparations and elicit an increased labeling of phosphatidate and phosphatidylinositol from 32Pi has been investigated. Two groups of brain muscarinic agonists are distinguished. Addition of acetylcholine, carbamylcholine, methacholine, or muscarine resulted in a 2-fold stimulation of phosphatidate and phosphatidylinositol labeling, while bethanechol, pilocarpine, arecoline, and oxotremorine were less effective. Simultaneous addition of two agonists from the more effective group did not result in any further increase in stimulated labeling, while the addition of agonists from the less effective group antagonized the stimulatory effect of carbamylcholine. All of the agonists could completely displace binding of [3H]quinuclidinyl benzilate, a muscarinic antagonist. The displacement of the labeled antagonist by the more effective agonists was more complex than that predicted from a simple mass action isotherm and was compatible with the interaction of the agonists with high and low affinity forms of the receptor. Conversely, the displacement data from less effective agonists did not deviate markedly from those predicted for interaction of the agonists with a single affinity form of the receptor. Dose-response curves for stimulated phosphatidate labeling obtained in the presence of acetylcholine, carbamylcholine, and methacholine were predominantly correlated with occupation of the low affinity form of the muscarinic receptor. These results suggest that the enhancement of phosphatidate and phosphatidylinositol turnover in brain is caused by agonist-mediated conformational changes in the muscarinic receptor and that the ability of an agonist to induce this conversion may be predicted by its differential binding to the high and low affinity forms of the receptor.