Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

Roles of 5-hydroxytryptamine and dopamine as neurotransmitters eliciting release of erythrophorotropic hormones in the fiddler crab,Uca pugilator

This article reviews the endocrinological, pharmacological and biochemical evidence ascribing neurotransmitter roles for 5-hydroxytryptamine (5-HT, serotonin) in eliciting the release of red pigment-dispersing hormone (RPDH) and for dopamine (DA) in stimulating the release of red pigment-concentrating hormone (RPCH) in the fiddler crab, $\text{Uca pugilator}$. 5-HT produces red pigment dispersion in intact crabs, but only indirectly. Likewise, DA evokes red pigment concentration $\text{in vivo}$ but it has no effect on red chromatophores (erythrophores) in isolated legs. The data obtained with 5-HT and DA agonists and antagonists on red pigment translocation $\text{in vivo}$ and $\text{in vitro}$, are consistent with their neurotransmitter candidacies in evoking the release of these erythrophorotropic hormones.     

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

Agonist-specific reverse regulation of muscarinic receptors by transition metal ions and guanine nucleotides

Transition metal ions, e.g. Mn²⁺, Ni²⁺ and Co²⁺ enhance $\text{in vitro}$ agonist binding to muscarinic receptors in mouse cortex or hippocampus. This effect arises mainly from the conversion of low to high affinity binding sites. Binding properties of antagonists in these brain areas, as well as those of both agonists and antagonists of medulla-pons muscarinic receptors, are insensitive to these ions. The induced interconversion can be reversed by either of the following procedures: (i) removal of the ions; (ii) thermal exposure; (iii) addition of micromolar concentrations of guanine nucleotides.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

DPI, a supposed selective agonist of inhibitory dopamine receptors, strongly increases rat diuresis through alpha-adrenergic receptor activation

The diuretic effect of dopamine (DA) was compared with that of other DA receptor stimulants in rats. While apomorphine (APO) and (±)N-n-propyl-norapomorphine (NPA) failed to increase urine excretion, (3, 4-dihydroxyphenylamino) -2-imidazoline (DPI), a supposed st$\text{i}$mulant of DA inhibitory receptors, exerted a far greater diuretic e$\text{f}$fect than DA itself. The diuretic effect of DA and that of DPI were antagonized by a-adrenergic receptor antagonists such as phentolamine and yohimbine and by bulbocapnine. In contrast, DA receptor blockers such as haloperidol, pimozide, sulpiride and ergometrine were totally ineffective. It is concluded that the diuretic effect of DA and DPI are mediated by stimulation of a-adrenergic receptors.     

Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Binding of the alpha adrenergic antagonists (³H) prazosin and (³H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (³H) prazosin and (³H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order $\text{epinephrine}\text{>}\text{norepinephrine}\text{a}\text{?}\text{isoproterenol}$, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (³H) prazosin and (³H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (³H) yohimbine specific binding. The affinity of prazosin for the (³H) yohimbine binding site is approximately 2500 times less than for the (³H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (³H) yohimbine than (³H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (³H) yohimbine binding sites in different arteries vary about two fold whilst for (³H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha₁ and alpha₂ adrenoceptors.     

Pharmacological effects of a specific dopamine D-1 antagonist SCH 23390 in comparison with neuroleptics

Neuroleptics such as thioxanthenes (cis($\text{Z}$)-flupentixol and cis($\text{Z}$)-clopenthixol) and phenothiazines (fluphenazine and perphenazine), which block both dopamine (DA) D-1 and D-2 receptors and the butyrophenones (haloperidol and spiroperidol), which block D-2 receptors only, are equipotent both behaviorally and clinically. A new compound SCH 23390 which selectively blocks DA D-1 receptors, resembles many neuroleptics in its pharmacological profile: antistereotypic effects in mice, rats and dogs, cataleptogenic effect and inhibitory effect on amphetamine circling. In contrast SCH 23390 has no effect on apomorphine-induced vomiting in dogs and little effects on 6-OHDA-denervated supersensitive DA receptors, stimulated by the DA agonist 3-PPP. In a series of experiments where methylphenidate-induced stereotyped gnawing in mice was inhibited by neuroleptics, it was shown that concomitant treatment with scopolamine or diazepam attenuated the effect of butyrophenones (D-2 antagonists). The same treatment attenuated the effect of phenothiazines, to a lesser extent, and hardly attenuated the effect of thioxanthenes and SCH 23390 at all. It is concluded that DA D-1 receptors are as important as D-2 receptors for the expression of neuroleptic activity in most animal models believed to be predictive of antipsychotic and extrapyramidal side-effect potential. However, the D-1 antagonist is less sensitive than D-2 antagonists to antimuscarinic compounds and benzodiapines.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

Conformational preferences of dopamine analogues for inhibition of norepinephrine N-methyltransferase. Conformationally defined adrenergic agents. 7

A series of analogues of dopamine (DA) with varying degrees of conformational flexibility have been examined as potential substrates or competitive inhibitors of the enzyme norepinephrine N-methyltransferase (NMT). A conformationally defined (rigid) analogue of the fully extended conformation of DA, 2-amino-6, 7-dihydroxybenzonorbornene hydrobromide ($\text{3}$; 6, 7-D2HX) proved to be a better substrate than the non-catechol parent 2-aminobenzonorbornene ($\text{4}$; 2HX). However, analogues $\text{3}$ and $\text{4}$ displayed equivalent competitive inhibitory activity toward phenylethanolamine (PEA). Neither 6, 7-ADTN (5), a DA analogue in the 2-aminotetralin (2AT) system, nor 6, 7-DTHIQ ($\text{7}$), a DA analogue in the tetrahydroisoquinoline (THIQ) system, showed substrate activity; 6, 7-ADTN was a poorer competitive inhibitor than the parent 2AT but 6, 7-DTHIQ was a better competitive inhibitor than its parent, THIQ ($\text{8}$). A tricyclic conformationally defined analogue $\text{9}$ of 6, 7-ADTN was devoid of either substrate or inhibitory activity. From these results it may be concluded that a fully extended side chain conformation is required for NMT substrate activity, and the better substrate activity for 6, 7-D2HX compared to $\text{4}$ is consistent with a proper catechol orientation for interaction with the norepinephrine (NE) binding site of NMT.     

LSD and related drugs as DA antagonists: Receptor-mediated effects on the synthesis and turnover of DA

We have earlier shown that d-lysergic acid diethylamide, LSD and its 2-bromo derivative, BOL like the dopamine (DA) antagonists haloperidol increased the rate of the $\text{in vivo}$ tyrosine hydroxylation in the striatum measured as the accumulation of DOPA after decarboxylase inhibition.Now we have found that several agents structurally similar to LSD increase the $\text{in vivo}$ tyrosine hydroxylation in the striatum. Psilocybin (50 mg/kg i.p.) and N,N-dimethyltryptamine (50 mg/kg i.p.) caused a short-lasting increase of DOPA accumulation, while mescaline (10 – 100 mg/kg i.p.) did not increase the DOPA accumulation. A marked increase of DOPA accumulation was observed after the 5-hydroxytryptamine (5-HT) antagonist cyproheptadine. The effects of LSD and structurally related drugs on the DOPA accumulation in the striatum appear to be mediated via DA antagonism at receptor level. However, these agents may control the DOPA accumulation via other receptors than DA receptors e.g. 5-HT receptors. A control of DOPA accumulation via receptors other than DA receptors appears to be predominant after treatment with N,N-dimethyltryptamine or psilocybin.     

Analysis of the inhibitor binding to the nucleoside site of phosphorylase b

The binding of inhibitors to site I of rabbit muscle phsphorylase $\text{b}$ has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both $\text{Δ}\text{H}$ and $\text{Δ}\text{S}$ of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase $\text{b}$ is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive $\text{T}$ conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active $\text{R}$ conformation, whereas non-exclusive binding of the activator was observed when caffeine was added.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Binding of opiates and endogenous opioid peptides to neuroleptic receptor sites in the corpus striatum.

Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with ³H-spiroperidol for common binding sites in rat striatum $\text{in vitro}$ (IC₅₀ in the range between 10^?6 and 10^?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since $\text{in vivo}$ studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites $\text{in vitro}$ and their potency to evoke “bizarre behavior” in rats $\text{in vivo}$. In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites $\text{in vitro}$ (IC₅₀ greater than 10^?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites.     

Kinetics of calcium efflux and exchange from Rana pipiens oocytes immediately following reinitiation of the first meiotic division: Comparison of various meiotic agonists and antagonists

The reinitiation of the meiotic divisions and the release of ⁴⁵Ca from the $\text{Rana}$$\text{pipiens}$ oocyte has been studied as a function of meiotic agonists and antagonists. Each of the meiotic agonists tested (progesterone, insulin, D-600, La³⁺) caused a decreased ⁴⁵Ca uptake and an increased efflux during the first 15 min after exposure. The effects of progesterone, D-600, and La³⁺ are not additive and progesterone will not release additional ⁴⁵Ca in oocytes pretreated with D-600 or La³⁺. Tetracaine inhibits both progesterone-induced release of ⁴⁵Ca and an early step in meiosis (nuclear membrane breakdown). [Tetracaine]_o required for 50% inhibition of nuclear breakdown decreases with decreasing [progesterone]_o suggesting competitive inhibition. The Ca, Mg-ionophore A23187 shows a similar competitive inhibition of progesterone-induced nuclear breakdown and stimulates a rapid release of ⁴⁵Ca within the first 1–3 minutes after exposure to the ionophore. Unlike progesterone, insulin, D-600, or La³⁺, the ionophore A23187 stimulates both uptake and efflux of ⁴⁵Ca by oocytes. These results suggest that both a reduced influx and a selective release of calcium from specific membrane sites is essential for steroid reinitiation of the meiotic divisions in $\text{R.}$$\text{pipiens}$ oocytes.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Effects of progesterone on the binding of estradiol-receptor to rabbit uterine chromatin

Three day progesterone treatment of ovariectomized rabbits increased $\text{in}$$\text{vitro}$ uterine estrogen-receptor binding to uterine chromatin. The increased binding was traced to changes in chromatin but not the cytosol. Both the number of chromatin acceptor sites and the binding affinity were higher in treated animals. Furthermore, chromatin acidic protein to DNA ratios from treated rabbits were higher by approximately the same factor as for binding sites. A mechanism of synergistic interaction is suggested.     

Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.