Maturation of the adrenal medulla—III: Practical and theoretical considerations of age-dependent alterations in kinetics of incorporation of catecholamines and non-catecholamines

Rats were sacrificed at 10-day intervals from birth to 50 days of age and the kinetics of uptake of epinephrine (E) and metaraminol (MA) into isolated adrenal medullary storage vesicles were measured. The K_m for epinephrine remained at about 30 μM throughout development, but the maximal uptake (U_max) at birth was 30 nmoles/30 min/100 μg of endogenous catecholamines compared to 2 nmoles at subsequent ages. The K_m for MA was about 4 mM at birth, 0·7 mM at 10–20 days of age and 1·2 mM thereafter, while U_max was approximately 170 nmoles initially, 50 nmoles at 10–20 days and 70 nmoles thereafter. The preference of the vesicles for uptake of E vs MA (both at 0·1 mM) was approximately 6 to 1 at birth, 1 to 1 at 10 days, 3·5 to 1 at 20 days and 5 to 1 thereafter, while the preference for E vs tyramine was 2 to 1, except at 10 days, when the ratio was 1 to 1. These data suggest that the two vesicular uptake mechanisms, typified by incorporations of E and of MA, develop independently of each other and that the age-dependent alterations are present both at the level of the vesicle membrane and intravesicular storage. The development of a kinetic model aids in the identification of probable sites of specific age-dependent alterations. Maturational changes in uptake and specificity can produce parallel changes in synthesis of catecholamines as determined by conversion of newly incorporated tyramine to octopamine.     

Inhibition of epinephrine and metaraminol uptake into adrenal medullary vesicles by aralkylamines and alkylamines

Two amine uptake mechanisms appeared to operate in isolated adrenal medullary storage vesicles; one site had a high affinity for epinephrine (K_m ～- 30 ωM) and low capacity (U_max ～- 20 nmoles/100 ωg of endogenous catecholamines), while the other had a low affinity $\text{(K}{}_{\mathrm{m}}\text{? 2}\text{mM}\text{)}$ and a higher capacity (U_max ? 130 nmoles). The low affinity site was non-specific and did not display competitive inhibition by agents which affected the high affinity, stimulated transport system. The high affinity system was inhibited in a purely competitive fashion by a variety of indoleamines and phenethylamines. but the two classes of compounds displayed different structure-activity relationships. Substitution on the α-carbon decreased the abilities of indoleamines to inhibit stimulated epinephrine uptake, but enhanced activity of phenethylamines. Ring hydroxylation reduced, and methoxylation eliminated, the inhibitory activity of tryptamine, but the same substituents markedly enhanced the activity of phenethylamines. Studied of compounds with restricted side-chain conformation indicated that a condensed structure favored activity in indoleamines, while an extended chain enhanced inhibition by phenethylamines. Linear alkylamines of 5- or 6-carbon length were also able to inhibit active epinephrine uptake. None of the agents inhibited the non-stimulated uptake component of metaraminol, which uses primarily the low affinity system. These data suggest that while indoleamines and phenethylamines do compete with epinephrine for attachment to the high affinity transport site in the vesicle membrane, the point of interaction is probably solely at the locus which binds the amine nitrogen; the remainder of the two types of molecule probably bind to at least two different sites adjacent to the N-binding area.     

Adrenal medullary storage vesicles of the spontaneously hypertensive rat.

The adrenal medullary vesicles of normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR) were examined in order to determine whether increased sympatho-adrenal activity plays a role in the elevation of blood pressure in the SHR. The uptake of ¹⁴C-epinephrine/100 μg of endogenous catecholamines in isolated SHR vesicles was higher than in NWR, while the uptake of ³H-metaraminol was the same as in NWR; thus, SHR vesicles exhibited a higher preference for epinephrine vs metaraminol compared to NWR. The difference in uptake was due to a lower K_m for epinephrine in SHR. The storage of amines was the same in SHR and NWR, as demonstrated by measurements of catecholamine to ATP ratios in purified vesicles, and effluxes from the vesicles of endogenous and newly incorporated amines. The ratio of catecholamines to dopamine β-hydroxylase (DBH, a marker for storage vesicles) was higher in SHR for three reasons: (1) there were fewer vesicles per gland; (2) there was less DBH per vesicle, indicated by an increased precursor/product ratio; and (3) there was a higher catecholamine content per vesicle, as shown by an increase in the ratio of heavy to light vesicles on discontinuous sucrose density gradients. SHR adrenals were depleted of catecholamines after insulin administration to a greater extent than were NWR adrenals, and both SHR and NWR exhibited induction of tyrosine hydroxylase and dopamine β-hydroxylase after insulin. None of these findings is consistent with the view that sympathoadrenal hyperactivity occurs in the SHR; the data suggest that hypoactivity occurs, perhaps secondarily to the hypertension.     

A quantitative study of the γ-aminobutyric acid (GABA) dose/conductance relationship at the lobster inhibitory neuromuscular junction

GABA-evoked changes in membrane conductance of lobster muscle fibres were measured using the ‘short’ cable equations. In 79 fibres, the mean resting conductance $\text{(?}{}_{\mathrm{m}}\text{L)}$ was 12.9μmho (S.D. ± 5.6 μmho). In the presence of GABA (5 μM to 640 μM) the membrane conductance increased with little change in resting potential and with no visible decline in response during exposure. In contrast with the non-cumulative GABA dose/conductance curves, cumulatively established curves attained an apparent maximum at GABA concentrations exceeding 80 μM.The non-cumulative dose/conductance curve was better fitted by a two independent binding-site receptor model than by a two-site high facilitation model or a single-site model. Values for the apparent GABA/receptor dissociation constant estimated by ‘least squares’ analysis (on the basis of the independent model) were similar to those estimated by double reciprocal plot (ca. 30 μM).     

Transport of dibromosulphthalein by isolated rat hepatocytes

Transport characteristics for uptake and release of dibromosulphthalein (DBSP) by isolated rat liver cells were studied. The rate of uptake is dependent on the extracellular protein concentration and follows Michaelis—Menten kinetics with respect to the unbound substrate concentration; the process has an apparent K_m of 2.1 ± 0.3 μM and a V_max of 2.0 ± 0.4 nmol/min per 10⁶ hepatocytes. The activation energy amounts to 109 ± 8 kJ/mol at 1 μM DBSP. Uptake is only partly dependent on metabolic energy and is independent of the Na⁺ gradient across the membrane. Adsorption to the cell membrane occurs with two types of binding sites with affinity K₁ = 1.3 μM; n₁ = 1.6 nmol/10⁶ cells and K₂ = 43 μM; n₂ = 3.9/10⁶ cells. The uptake is inhibited by indocyanine green and evans blue.The rate of release of DBSP is independent of the extracellular protein concentration and follows Michaelis-Menten kinetics with an apparent K_m = 21 ± 4 nmol/10⁶ hepatocytes. Release is independent of the Na⁺ gradient across the membrane and is only slightly dependent on metabolic energy. Omission of Ca²⁺ from the incubation medium did not have any influence on the uptake rate of DBSP but lowered the rate of release from the cells by about 10%. It is concluded that the uptake of DBSP into rat hepatocytes occurs against an electrochemical gradient in contrast to the release of DBSP from the cells. The release process shows characteristics similar to biliary secretion in vivo, its capacity under first order kinetic conditions is a factor of 100 lower than that of the uptake process.     

Detoxification/toxification of cadmium in scorpionfish (Scorpaena guttata): Acute exposure

Scorpionfish were exposed to sea water (control), sea water dosed with 25 mg Cd/l (0.4 96-h LC₅₀), and 50 mg Cd/l (0.8 96-h LC₅₀) as CdCl₂ for 96 h. These fish were then analyzed to determine the effects of near-lethal Cd exposure on mechanisms of detoxification by metallothionein and the potential for toxification of enzymes in several tissues. In scorpionfish exposed to 50 mg Cd/l, the highest concentrations of metallothionein pool Cd occurred in liver ($\text{532 ± 68 μ}\text{mol/wet}\text{kg; mean}\text{±}\text{sd}\text{; n=3}$) followed by intestine (151 ± 55 μmol/wet kg), gills (27.1 ± 9.6 μmol/wet kg) and then kidney (26.8 ± 6.1 μmol/wet kg). In these same fish, the highest concentrations of enzyme pool Cd occurred in kidney (65 ± 41 μmol/wet kg), followed by gills (33.4 ± 2.2 μmol/wet kg), intestine (21 ± 12 μmol/wet kg) and then liver (3.9 ± 1.6 μmol/wet kg). Based upon this partitioning of Cd, the order of sensitivity of tissues, at near-lethal Cd concentrations, would appear to be kidney > gills > intestine > liver.     

Metabolic rate of phenacetin and of paracetamol in dogs before and after treatment with phenobarbital or skf 525 A

In six male mongrel dogs the apparent biological half life ($\text{t}\text{2}$) in plasma of intravenously injected phenacetin decreased from 34·6 to 27·2 min after treatment with phenobarbital (25 mg/kg/day for 8 days) (P < 0·025). In the same dogs, the apparent $\text{t}\text{2}$ of intravenously injected phenazone decreased from 78·6 to 32·4 min (P < 0·05). Treatment with SKF 525 A (25 mg/kg) increased the $\text{t}\text{2}$ of phenazone in three other dogs from 70·6 to 246·7 min, whereas the $\text{t}\text{2}$ of phenacetin remained unaffected.The concentration of phenacetin at zero time of intravenous injection increased from 29·5 ± 5·9 S.E. to 43·5 ± 12·6 μg/ml plasma (P < 0·2) after phenobarbital treatment; pretreatment with phenobarbital, however, had no influence on the mean concentration of phenazone at 0 time. SKF 525 A did not influence the zero time concentration of either phenacetin or phenazone.Beagle liver microsomal protein and cytochrome P-450 concentrations increased from 3·2 to 4·2 mg/ml and from 1·0 to 4·1 n-mole/mg protein, respectively, after phenobarbital treatment. Treatment with phenobarbital had no influence on the rates of formation of paracetamol and p-phenetidin nor was the apparent K_m of phenacetin affected.The binding of phenacetin to blood constituents increased from 50 per cent at 5 μg/ml to 59 per cent at 1 μg phenacetin/ml. In the presence of 30 μg phenobarbital/ml blood, the binding of phenacetin at 1 μg/ml blood decreased by 22 per cent.It is concluded that the decrease of the apparent $\text{t}\text{2}$ of phenacetin in dog plasma after phenobarbital treatment may be due to a change in the apparent volume of distribution and/or to some stimulation of an NADPH-dependent enzyme system of the liver less affected by treatment with SKF 525 A than in other species.     

Dimethyl sulfoxide: Inhibition of acetylcholinesterase in the mammalian heart

The heart rate of the isolated, perfused, working rat heart was significantly and equally depressed by 1 × 10^?6 M acetylcholine (ACh) and by 6 × 10^?5 M 4-ketoamyltrimethylammonium (4K), a cholinomimetic agonist. Dimethyl sulfoxide (DMSO) (10 μl/ml, 140 mM) strongly potentiated the effect of ACh but did not alter the effect of 4K. DMSO (10 μl/ml, 140 mM final concentration) alone had no significant effect upon heart rate when added to the perfusate in incremental additions of 1 μl · (ml perfusate)^?1 · min^?1 over a 10-min period. The specific activity of atrial homogenate cholinesterase was $\text{48.8 ± 3.46}\text{nmoles}\text{·}\text{min}{}^{\mathrm{?1}}\text{· (}\text{mg protein}\text{)}{}^{\mathrm{?1}}\text{(}\text{mean}\text{±}\text{S.E.M.}\text{), 38.2 ± 1.60}$ for butyrylcholinesterase, and 11.2 ± 0.86 for acetylcholinesterase (AChE). True AChE activity (measured in the presence of a maximally effective concentration of tetraisopropylpyrophosphoramide) had a V_max of 13.4 ± 0.17 nmoles · min^?1 · (mg protein)^?1 and an apparent K_m value of 1 × 10^?4 M acetylthiocholine. At this K_m substrate concentration, DMSO inhibited atrial AChE activity (I₅₀ = 9 μl/ml). At the concentration tested, DMSO inhibited atrial AChE and potentiated ACh effects.     

The fate of the ethanol analogue 1,3-butanediol in the dog. An in vivo-in vitro comparison

In view of the current interest in 1,3-butanediol as food additive or potential drug its pharmacokinetics have been investigated in the dog and compared to its oxidation in vitro by alcohol dehydrogenase. Plasma disappearance of i.v. doses of 5.5 mmol/kg were zero order followed by first order. Assuming Michaelis-Menten kinetics a V_max of 1.23 ± S.D. 0.27 μmol/min/g of liver and a K_m of 1.15 ± 0.85 mM could be calculated. The corresponding values for 1,3-butanediol metabolism by alcohol dehydrogenase in vitro were 1.62 ± 0.34 μmol/min/g of liver and 5.11 ± 1.45 mM. Hepatic vein catheterizations were used to measure hepatic blood flow (18.1 ± 2.8 ml/min/kg) and the fraction of butanediol disappearing in the liver, which was only 34.2 ± 6.6 per cent. Compared to ethanol, V_max of 1,3-butanediol was 15 per cent smaller in vitro, 45 per cent smaller in vivo, K_m was 3 times larger in vitro and 60 per cent smaller in vivo. The splanchnic elimination fraction of 1,3-butanediol was about $\text{1}\text{2}$ the one of ethanol. These data are consistent with the concept, that oxidation by alcohol dehydrogenase is the major route of butanediol elimination. The differences between 1,3-butanediol and ethanol metabolism, however, render different pharmacological and toxicological effects likely.     

Clinical applicability of current pharmacokinetic models: Splanchnic elimination of 5-fluorouracil in cancer patients

What can be inferred from limited clinical data by using current models of hepatic elimination? We examined this question by analyzing previously published data on the steady-state uptake of the anticancer agent 5-fluorouracil (5-FU) in seven cancer patients in terms of the venous equilibration model, the undistributed and distributed forms of the sinusoidal perfusion model, and the convection-dispersion model. Because of appreciable extrasplanchnic removal of 5-FU, the value of the steady infusion rate was not used in our analysis. When the data from all patients were pooled by plotting the measured hepatic venous concentration against the measured hepatic arterial concentration, the high concentration data fell on a limiting straight line of slope 1, indicating that at high dose rates elimination of 5-FU in both the liver and gastrointestinal tract was close to saturation. The intercept of this line gave a model-independent estimate of V_max/Q= 48.0±11.6 (SD) μM for the pooled data set, where V_max is the maximum splanchnic elimination rate of 5-FU, and Q is the hepatic blood flow. The low concentration data points fell on a limiting straight line through the origin, from which model-dependent values of the Michaelis constant were determined. The venous equilibration model gave K _m=9.4μM,while the undistributed sinusoidal perfusion model gave K _m ^* =26,5μM. With these values of K _m,both models fit the pooled data equally well. These methods were applied to analyses of the five individual data sets which contained sufficiently high concentration data points. The resulting mean values were V_max/Q=41.0±5.1 (sem) μM,K _m=8.4±1.3μM and K _m ^* =23.2±3.2 μM. However, the splanchnic region is a highly heterogeneous organ system, for which an undistributed analysis provides no more than an upper bound on the Michaelis constant K _m ⁺ (K _m ⁺ ?K _m ^* ).A perfusion model distributed to represent total splanchnic elimination is developed in the Appendix. Using previous estimates of the degree of functional heterogeneity in the liver alone, this model yields K _m ⁺ values for individual patients which have a mean of 20.3±2.8 μM.     

Two systems are involved in the sulfobromophthalein uptake by rat liver cells: One is shared with bile salts

It is generally believed that the uptake of sulfobromophthalein by rat liver cells is mediated by a single carrier. Accordingly, kinetic plots obtained using a wide concentration range, failed to give any evidence of heterogeneity in the uptake and gave a K_mof 8.9 ± 2.5 μM and a maximal velocity of 4.2 ± 0.8 nmoles/min/10⁶ cells. Na⁺-taurocholate inhibited competitively, but only partially, sulfobromophthalein uptake by isolated rat liver cells. The taurocholate sensitive component of sulfobromophthalein uptake followed Michaelis Menten kinetics with a K_mof 0.9 μM and a V_max of 0.47 nmole/min/10⁶ cells. Taurocholate inhibition reveals an heterogeneity in the uptake of sulfobromophthalein not shown in classical kinetic plots, indicating that two carriers are involved in the uptake of this dye by rat liver cells. The taurocholate sensitive carrier which displays an affinity for sulfobromophthalein ten times higher than that of taurocholate insensitive one, is probably identical to the Na⁺-independent carrier involved in bile salt uptake.     

Effects of thiamin antagonists on drug hydroxylation and properties of cytochrome P-450 in the rat

The administration of small amounts of thiamin (0.3 $\text{μg}\text{day}$ or more, i.p., for 21 days) depressed musomal cytochrome P-450 content and the V_max of aniline hydroxylase when compared to values obtained from rats fed a thiamin-deficient diet (approximately 0.1 μg of thiamine/day in basal diet). The concurrent administration of neopyrithiamin (50 $\text{μg}\text{day}$, i.p.) eliminated the depressant effect of 10.0 μig of thiamin/ day, but was without significant effect in rats receiving more than 100 μg of thiamin/ day. In contrast, 100 μg of oxythiamin/day had no thiamin-opposing effect on cytochrome P-450 content and only partially counteracted the effects of 1 μg of thiamin/day on aniline hydroxylase activity. These treatments were without significant effect on the K_m for this reaction. Using ethyl isocyanide as the ligand, there appears to be a qualitative change induced in the cytochrome P-450 from thiamin-deficient rats. The absorption peak height ratios indicate that cytochrome P₁-450 is increased in a manner analogous to that produced by the administration of 3-methylcholanthrene. This was supported by the fact that aniline binding, as evidenced by increased ΔA_max, is enchanced in musomes from thiamin-deficient animals, whereas the hexobarbital spectral shift was unaltered.     

Anticonvulsant and proconvulsant properties of a series of structural isomers of piperidine dicarboxylic acid

Anticonvulsant and convulsant effects of various piperidine dicarboxylic acids have been evaluated following their intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) injection in DBA/2 mice, a strain of mice genetically susceptible to sound-induced seizures.Protection against sound-induced seizures occurred after intraventricular administration of (±)cis-2,3-piperidine dicarboxylic acid (0.017–0.045 μmol), (±)trans-2,3-piperidine dicarboxylic acid (0.018–0.33 μmol) and (±)cis-2,4-piperidine dicarboxylic acid (0.57–1.68 μmol).Protection against sound-induced seizures occurred after intraperitoneal injection of (±)cis-2,3-piperidine dicarboxylic acid (0.52–1.8 mmol/kg).Myoclonus or convulsions occurred at various times after the intraventricular injection of cis-2,3-piperidine dicarboxylic acid, trans-2,3-, cis-2,4-, cis-2,5- and cis-2,6-, piperidine dicarboxylic acids, and after the intraperitoneal injection of trans-2,3-piperidine dicarboxylic acid. The latter effect was blocked by pretreatment with 2-amino-7-phosphonoheptanoic acid (0.33 mmol/kg, i.p.) a potent and specific antagonist of excitation induced by $\text{N-}\text{methyl-}\text{d}\text{-aspartate}$.The anticonvulsant action of cis-2,3-piperidine dicarboxylic acid and the convulsant action of trans-2,3-piperidine dicarboxylic acid were associated with predominant antagonist and agonist actions respectively, at receptors preferring $\text{N-}\text{methyl-}\text{d}\text{-aspartate}$.     

Reversible swelling evoked by epinephrine, 8-bromo-cyclic 3',5'-guanosine monophosphate and feeding in the mast cells of the rat

Swelling, shape distortion, and sectorially decreased granule population-density can be observed in rat-mesentery mast cells exposed to epinephrine in vivo or in vitro. To a lesser degree, such changes are also noted in mast cells of untreated rats. Changes due to epinephrine were quantified both in mesentery spreads and in fixed and photographically magnified isolated rat peritoneal-fluid mast cells. Threshold effectiveness of epinephrine was 0.5 μg/kg in vivo (i.v.) and 10^?8 moles/liter in vitro. Rapidity, partly linear log dose-response relationships, and reversibility characterize the responses of mast cells to epinephrine. Within limits, re-exposure to epinephrine of spontaneously deactivated mast cells resulted in renewed change. N⁶-2′-O-Dibutyril-cyclic 3′,5′-adenosine monophosphate (diBu-cAMP) inhibited, and 8-bromo-cyclic 3′,5′-guanosine monophosphate (8-Br-cGMP) reproduced, the effects of epinephrine; like the catecholamine, neither cyclic nucleotide derivative caused a release of histamine or a degranulation of mast cells in the manner that compound $\text{48}\text{80}$ was shown to do. A return to normality of mesenteric mast cells of $\text{48}\text{80}\text{-}\text{treated}$ rats required between 24 and 48 hr; in the epinephrine-treated rat, this return was complete within 60 min. Feeding significantly increased the number of morphologically altered mast cells present in mesenteries of 24 hr-fasted rats. A renewed fast reduced altered mast cell numbers to prefeeding levels. Although mast cell alterations evoked by feeding appeared to be morphologically similar to those caused by epinephrine or 8-Br-cGMP, the catecholamine cannot be the mediator of such changes because phenoxybenzamine, an α-adrenergic blocking agent, prevented the effect of epinephrine but not that of feeding on mast cells.     

Reversion by vitamin K of aflatoxin B1 (AFB)-induced inhibition of oxygen uptake in three AFB-susceptible bacteria

The effect of Vitamin K, on the inhibition of oxygen uptake in Bacillus brevis (2611), Bacillus megaterium (1368) and Flavobacterium aurantiacum by aflatoxin B₁ (AFB) has been investigated.At 1.5 mM, vitamin K completely reversed oxygen inhibition by 10 $\text{μg}\text{ml}$ AFB in the three bacteria and by 50 $\text{μg}\text{ml}$ APB in B. brevis, the most susceptible of the three bacteria to AFB. Vitamin K did not reverse the inhibitory effect of 100 $\text{μg}\text{ml}$ AFB in any of the three bacteria except B. megaterium, neither did 1.5 mM 2,4-dinitrophenol (DNP) reverse the inhibition at 10 $\text{μg}\text{ml}$ and 100 $\text{μg}\text{ml}$ levels of AFB in any of the three bacteria.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.     

beta-adrenergic receptors of human leukocytes: studies with intact mononuclear and polymorphonuclear cells and membranes comparing two radioligands in the presence and absence of chloroquine

Owing to the large differences in reported values for β-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[³H]dihydroalprenolol (DHA) and $\text{(±)-}\text{[}{}^{125}\text{I]iodohydroxybenzylpindolol}$ (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10–24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000–60,000 sites/cell) of low affinity (K_d 12–15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 ,μM chloroquine not only in PMN cells but also in the lysome-poor MN cells (? 90% lymphocytes), leaving 2000–3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (±)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 μM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent K_d. Mean (± S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 ± 100 sites/MN cell and 1135 ±129 sites/PMN cell (K_d 143–153 pM) using (-)-DHA; and 1487 ± 210 sites/MN cell and 1065 ± 69 sites/PMN cell [avg. K_d(±) 224–274 pM] using (±)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the β₂-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (±)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (±)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[³H]DHA, 1 ,μM (-)-propranolol and 10, μM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for β-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature.     

Effect of vitamin D3 derivatives on calcium concentrations in the serum of rats treated with stannous chloride

Calcium concentrations in the serum of rats were significantly increased by treatment with vitamin D₃ (frsol|25 μg/100 g), but this increase was prevented by a single intraperitoneal administration of tin (Sn²⁺, $\text{3.0 mg}\text{100 g}$). The decrease in serum calcium levels after the administration of Sn²⁺ was reversed by treatment with synthetic 1α-hydroxyvitamin D₃ ($\text{0.25 μg}\text{100 g}$) or 1α,25-dihydroxyvitamin D₃ ($\text{0.25 μg}\text{100 g}$). These results suggest that the decrease in serum calcium levels produced by the administration of Sn²⁺ may be caused by inhibition of metabolism of 25-hydroxyvitamin D₃ to 1α,25-dihydroxyvitamin D₃ in the kidneys.     

The effect of alkylating agents on adenosine 3',5'-monophosphate metabolism in Walker carcinoma.

The effect of some alkylating agents on the activity of the enzymes adenylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase has been studied using Walker carcinoma cells in tissue culture. The monofunctional agent 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), which has previously been shown to elevate the level of adenosine 3′,5′-monophosphate (cyclic AMP) in sensitive Walker cells, has been shown to have no effect on the activity of adenylate cyclase either in the presence or absence of the protecting agent 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl-AlC). Chlorambucil (p-N,N(di-2-chloroethylamino)phenylbutyric acid) (5 μg/ml) while having no effect on either the basal or fluoride-stimulated adenylate cyclase activity caused an inhibition of the high affinity form of the cyclic AMP phosphodiesterase which reached a maximum after 1 hr. This was accompanied by an increase in the intracellular level of cAMP which was proportional to the dose of chlorambucil up to a maximal 2-fold increase at 6.4 μg/ml, a dose which caused complete inhibition of cell growth. Further increases in the concentration of chlorambucil up to 100 μg/ml caused no further increase in cAMP level. Merophan ($\text{dl}\text{-}\text{o}\text{-N,N(}\text{di}\text{-2-}\text{chloroethylamino}\text{)}\text{phenylalanine}$) (0.5 μg/ml) similarly caused an inhibition of the low K_m form of the phosphodiesterase, but the rate of inhibition was slower than that observed with chlorambucil. The molecular forms of the cAMP phosphodiesterase in Walker cells sensitive or resistant to chlorambucil have been resolved using Sepharose 6B gel chromatography. The resistant lines displayed a reduction in the specific activity of the high affinity form of the enzyme which was accompanied by a shift to lower molecular weight forms. This could explain the lack of effect of chlorambucil on cAMP levels in Walker cells with acquired resistance to this agent.