Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Prostaglandin F2α receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents

Various enzymes and proteins reagents inhibited [³H]prostaglandin F₂α binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components.Preincubation of membranes with phoshpolipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and $\text{N-}\text{ethylmaleimide}$ resulted in moderate to drastic inhibitions of [³H]prostaglandin F₂α binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for $\text{N-}\text{ethylmaleimide}$), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca²⁺ was not required for its inhibition of [³H]prostaglandin F₂α binding. Protein reagents inhibition of binding was differently influenced by added Ca²⁺ i.e., loss of binding increased with some ($\text{N-}\text{ethylmaleimide}$), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca²⁺ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups.Treatment of membranes wiht trypsin and $\text{N-}\text{ethylmaleimide}$ selectively abolished high affinity prostaglandin F₂α receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzen and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F₂α receptors or by severely reducing their affinities.The occupancy of receptors by prostaglandin F₂α afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.     

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.     

Adriamycin inactivates cytochrome c oxidase by exclusion of the enzyme from its cardiolipin essential environment

The regulation of ligand binding to the muscarinic acetylcholine receptor in developing chick heart has been studied using the radiolabeled antagonist [³H]quinuclidinyl benzilate (QNB). In assays containing only buffer and a source of receptor protein, the antagonist radioligand bound to a single, high affinity state of the receptor. If Mg²⁺ and EDTA were added, [³H]QNB bound to a single, low affinity state. The guanine nucleotide analog, guanylylimidodiphosphate [Gpp(NH)p], reversed the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ so that [³H]QNB again bound only to a single, high affinity state. Sodium could also reverse the effect of $\text{Mg}{}^{\mathrm{2+}}\text{EDTA}$ on antagonist binding but the effects of sodium and Gpp(NH)p on [³H]QNB binding were not additive.     

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.     

The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

The effects of CaCl₂, MgCl₂, LaCl₃ and some alkali halides on $\text{Bacillus subtilis}$ cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with $\text{B. subtilis}$ membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca²⁺, Mg²⁺ and La³⁺, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.     

Electron paramagnetic resonance study on calmodulin: conformational change and interaction with divalent cations

Biologically active spin labelled derivatives of calmodulin were prepared and used to study CA²⁺- and Mg²⁺-induced conformational changes of the protein. The rotational correlation time of the spin labelled residues increased upon addition of divalent cations. Two calcium ions per spin labelled calmodulin were found to induce a 75% conformational change, whereas four calcium ions were necessary for a maximum conformational change. The increase in rotational correlation time induced by Mg²⁺ is less pronounced. Two different covalently attached spin labels (iodoacetamide and maleimide) were compared and marked differences were found in their chemical stability. The binding of manganese ions to calmodulin could be observed directly from the electron paramagnetic resonance spectra of these paramagnetic ions. Two specific classes of binding sites, each binding two manganese ions with $\text{k}{}_{\mathrm{<mtext>D</mtext>}}\text{= 0.6 × 10}{}^{\mathrm{?6}}\text{m}\text{and}\text{k}{}_{\mathrm{D}}\text{= 3 × 10}{}^{\mathrm{?5}}\text{m}$, respectively, were determined. Further ion binding occurs at non-specific sites.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.     

Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by positron emission tomography

Positron Emission Tomography (PET) was used to analyse $\text{in vivo}$ antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. ¹¹C-MONB concentration was determined $\text{in vivo}$ in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB.The kinetics of binding of ¹¹C-MQNB were not the same $\text{in vivo}$ and $\text{in vitro}$. The apparent dissociation rate of ¹¹C-MQNB $\text{in vivo}$ was much slower (by 1 to 2 orders of magnitude) than that observed $\text{in vitro}$ with ³H-QNB. After atropine injection, ¹¹C-MNQB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. ¹¹C-MQNB kinetics were analysed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, β-casomorphins, [D-Ala², D-Leu⁵] enkephalin and Sandoz peptide, FK 33–824, were examined by intracerebroventricular administration in rats. Their relative potencies $\text{in vivo}$ were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [³H]naloxone binding in the absence and presence of sodium ions for morphine (μ) receptors and against ¹²⁵I-[D-Ala², D-Leu⁵] enkephalin binding for enkephalin (δ) receptors. A good correlation between analgesic activity and morphine (μ) receptor but not enkephin (δ) receptor binding affinity was obtained. These data extend the hypothesis that morphine (δ) receptors mediate the major portion of the analgesic activity of opioids.     

Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells

We have studied muscarinic acetylcholine (ACh) receptors in intact atrial and ventricular heart cells dissociated from 8-day chick embryos and maintained in sparse cell cultures. Two specific antagonists, [³H]quinuclidinyl benzilate (QNB) and [³H]N-methyl scopolamine (NMS), bind to surface sites with affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{s}\text{? 40}\text{and}\text{400}\text{p}\text{M}$, respectively). The concentration of [³H]QNB sites in ventricular cell cultures (460 fmole/mg protein) was comparable to the concentration of sites in atrial cultures (420 fmole/mg protein). The same result was obtained with [³H]NMS. Autoradiography following incubation in saturating concentrations of [³H]QNB shows that nearly all of the atrial and ventricular myocytes were labeled and that the distribution of grains over individual cells was uniform. The mean binding site density was 109/μm² for atrial cells 117/μm² for ventricular cells. In contrast to the antagonist binding results, microelectrode recordings from individual myocytes or from small clusters of cells showed that many more atrial myocytes (89%) were sensitive to 10^?4M carbachol than were ventricular myocytes (26%). Saline extract of embryonic brain tissue added to the culture medium did not alter the number or distribution of ligand binding sites but it produced a 2.6-fold increase in the number of carbachol-sensitive ventricular cells.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

Identification and characterization of angiotensin II receptors in cardiac sarcolemma

We have used [¹²⁵I] angiotensin II to investigate the presence of specific angiotensin II receptors in beef heart sarcolemmal membranes. The observed binding is saturable, reversible and specific. The apparent equilibrium dissociation constant is 2.23 ± 0.15 ($\text{x}$ ± SEM) and the maximal number of binding sites per mg membrane protein is 32.8 ± 5.4 fmol ($\text{x}$ ± SEM). The specific binding is 80–100% of the total [¹²⁵I] angiotensin II bound and is directly proportional to membrane protein concentration over the range of 33–173 μg protein per ml. Angiotensin II and its antagonists competed for binding in a potency order of (agent, K_i): angiotensin II, 0.9nM > Sar¹ Ala³, 7 nM > Sar¹-Ile³, 51 nM > Sar¹-Leu³, 427nM > angiotensin I, 1709 nM. The ability to characterize and quantify these receptors should now provide a method for investigating the mechanisms underlying the effects of angiotensin II on myocardial tissues.     

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.     

Extracellular magnesium is not required for beta adrenergic drug-receptor interactions in intact human lymphocytes

We have investigated the effect of extracellular magnesium ions on the function of beta adrenergic receptors in $\text{intact}$ human lymphocytes. We examined adenylate cyclase stimulation by isoproterenol displacement of (-) (³H) dihydroalprenolol from beta receptor sites, and down regulation (desensitization) of beta receptors by prolonged exposure of the cells to isoproterenol. Contrary to results obtained using broken cell preparation, in none of these situations did the presence or absence of extracellular magnesium ions make any difference. The importance of selecting the most nearly physiological preparations for conducting $\text{in vitro}$ studies that may be extrapolated to the whole organism is stressed.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain

Receptors for the specific muscarinic radioligand [³H]quinuclidinyl benzilate ([³H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of [³H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized [³H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues.     

Kinetics of monensin complexation with sodium ions by 23Na NMR spectroscopy

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by ²³Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na⁺ + Mon^? ? MonNa. The dissociation rate constant at 25°C is 63 s^?1, with an activation enthalpy of 10.3 $\text{kcal}\text{mol}$ and activation entropy of ?15.8 $\text{cal}\text{mol}$ deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.