Receptor Dissociation Constant Research Articles

Opiate receptor avidity is reduced in non-motor impaired MPTP-lesioned rhesus monkeys

Opiate receptor avidity, roughly equivalent to the ratio of unoccupied receptor density to the receptor dissociation constant ( B′ max/ K D), was measured in four MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned rhesus monkeys and nine normal controls with positron emission tomography (PET) and 6-deoxy-6-β-[ 18 F ]fluoronaltrexone (cyclofoxy, CF), a μ- and κ-opiate receptor antagonist. Although the MPTP-lesioned monkeys were dopamine deficient as measured with [ 18 F ]- l-fluorodopa ([ 18 F ]-DOPA) and PET [Doudet et al., 6-[ 18 F ]- l-DOPA imaging of the dopamine neostriatal system in normal and clinically normal-MPTP-treated rhesus monkeys, Exp. Brain Res. 78 (1989) 69–80], they had clinically recovered from the acute motor effects of MPTP exposure. Opiate receptor avidity was found to be reduced by 30–35% in the opiate-receptor rich areas of caudate, anterior putamen, thalamus, and amygdala of the MPTP-lesioned animals. The results suggest that opiate pathways make a significant contribution to the adjustment of cortico–striatal–thalamic pathway activity and thereby to behavior in rhesus monkeys following dopamine loss.

NMDA Receptor Characteristics in the Brain of Fetal Guinea Pigs Following In Utero Hypoxia and Recovery. † 201

Previous studies have shown that in utero hypoxia results in brain cell membrane dysfunction and alters N-methyl-D-aspartate (NMDA) receptor characteristics in fetal guinea pigs. The present study tests the hypothesis that modification of brain cell membrane function during in utero hypoxia persists following recovery. Six pregnant guinea pigs at term were exposed to 7% oxygen for 1 hour. Brain tissue obtained from fetal guinea pigs immediately after in utero hypoxia was compared to that of fetuses exposed to hypoxia but allowed to recover in utero for 24 hours. ATP and phosphocreatine (PCr) levels were determined biochemically as indices of brain tissue energy metabolism. ATP and PCr levels were significantly higher in the recovery group compared to the hypoxic group(5.22±0.95 vs. 1.5±0.02 and 3.73±0.28 vs. 0.66±0.17 μ mol/g brain, respectively, p<0.01). Brain cell membrane Na+,K+-ATPase activity was measured as an index of brain cell membrane function. The recovery group demonstrated a significantly higher activity of Na+,K+-ATPase compared to the hypoxic group(35.1±4.0 vs. 25.2±7.0 μ moles Pi/mg protein/hr, p<0.05).3[H]MK-801 binding (2.5-50 nM) in the presence of glutamate and glycine was performed as an index of NMDA receptor modification. Receptor number(Bmax) in the recovery group was similar to the hypoxic group(1.28±0.04 vs 1.25±0.18 pmol/mg protein). In addition receptor dissociation constant (Kd) was similar in the two groups (5.61±0.30 vs 4.94±0.84 nM). This preliminary data show that following recovery from in utero hypoxia, brain cell energy metabolism and brain cell membrane function improve, but that hypoxia-induced alterations in NMDA receptor characteristics persist. We speculate that alterations in the NMDA receptor following in utero hypoxia may contribute to delayed brain cell injury despite recovery of cellular energy metabolism and cell membrane function.

Partial Knockdown of Gαi2Protein Is Sufficient to Abolish the Coupling of PYY Receptors to Biological Response in Renal Proximal Tubule Cells

Peptide YY (PYY)-preferring receptors are expressed in the renal proximal tubule cell clone Cl.10 isolated from the PKSV-PCT cell line. They mediate PYY-inhibited cAMP production through coupling with pertussis-sensitive Gi proteins. Previous GαiRNA antisense experiments demonstrated the exclusive coupling of the PYY receptor to the Gi2 protein. Here we characterized a clone stably expressing Gαi2antisense RNA which exhibited only a partial decrease in Gαi2content (#60 %) as estimated by Western blot. When compared to control Cl.10 cells, this clone, referred to as Cl.10(t), exhibited : (i) an increase in the dissociation constant of PYY receptors (6.42 vs 0.63 nM) ; (ii) a complete absence of inhibition of [125I]PYY binding by GTPγS and GTP ; (iii) the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels ; (iv) the failure of PYY to stimulate [35S]GTPγS binding to membranes. These findings show that partial knockdown of Gαi2expression in Cl.10 cells completely abolish the coupling of PYY receptors to biological response.

Gαi RNA Antisense Expression Demonstrates the Exclusive Coupling of Peptide YY Receptors to Gi2 Proteins in Renal Proximal Tubule Cells

A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M. (1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense G alpha i protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5'-noncoding region of G alpha i2 or G alpha i3 used as specific antisense templates. A Cl.10/alpha i2-clone was selected which displayed a drastic decrease (> 90%) of the expression of G alpha i2 without changes of G alpha i3, G alpha s, and G beta subunits (G alpha i1 is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of 125I-PYY binding by guanosine 5'-O-(thiotriphosphate) (GTP gamma S); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (> 80%) of G alpha i3 as compared to untransfected cells. The sensitivity to GTP gamma S and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to G alpha i2.

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH92]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg7]-vasopressin, [Thr4,Gly7]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile3]-pressinoic acid (tocinoic acid)). The dissociation constant (Kd) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), Kd (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, Kd values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (Bmax) on D0 (910.0 ± 25.1 fmol mg−1 protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg−1 protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg−1 protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, Bmax values were constant and similar to that on D5 of the early luteal stage. Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.

Receptors for atrial natriuretic peptide are decreased in the kidney of rats with streptozotocin-induced diabetes mellitus.

To determine whether decreased renal responsiveness to atrial natriuretic peptide (ANP) in diabetes is mediated by alterations in the renal ANP receptor, ANP receptor density and affinity were measured 17-20 d after streptozotocin injection and compared with values in vehicle-treated controls and streptozotocin-treated rats made euglycemic with insulin. Plasma ANP concentration was significantly greater in hyperglycemic diabetic rats than in control or euglycemic diabetic rats. Both in glomeruli and inner medulla, ANP receptor dissociation constant did not differ among the three study groups, whereas the maximum binding capacity was decreased significantly in hyperglycemic diabetics in comparison with controls and euglycemic diabetics. Glomerular clearance receptors were also decreased significantly in hyperglycemic diabetic rats in comparison with control and euglycemic diabetic rats. To determine whether the decreased number of renal ANP receptors in diabetic rats was associated with a decreased biological response, we measured ANP-dependent cyclic GMP (cGMP) accumulation by isolated glomeruli and inner medullary collecting duct cells in vitro. cGMP accumulation was significantly less in hyperglycemic diabetic rats than in controls or euglycemic diabetic rats both in the presence or absence of the phosphodiesterase inhibitor zaprinast. cGMP phosphodiesterase activity in inner medullary collecting duct cells obtained from control and hyperglycemic diabetic rats did not differ. Thus, the decreased number of biologically active ANP receptors in the kidneys of diabetic rats is accompanied by decreased biological responsiveness in vitro and provides a potential explanation for the reduction in renal sensitivity to ANP in this condition.

IGF-I and EGF receptors in the pigmented rabbit bulbar conjunctiva.

The purpose of this study was to investigate whether receptors for insulin-like growth factors I and II (IGF-I and IGF-II), insulin, epidermal growth factor (EGF), and transforming growth factor alpha (TGF alpha) are present on the apical surface of the pigmented rabbit bulbar conjunctiva. Binding of 125I-labelled ligands to the apical surface of the pigmented rabbit bulbar conjunctiva was conducted at 4 degrees C in the absence and presence of excess unlabeled ligands. There was no evidence for the existence of IGF-II, insulin or TGF alpha receptors in the isolated pigmented bulbar conjunctiva. Only IGF-I and EGF receptors appeared to be present. The apparent dissociation constant (Kd) of IGF-I receptors was 206 +/- 13 pM and that for EGF was 51 +/- 5 pM. The number of receptors per 95 mm2 of bulbar conjunctiva was (6.0 +/- 0.2)X10(9) for IGF-I and (2.4 +/- 0.1)X10(9) for EGF. There was no crossover binding of either IGF-I or EGF to one another's receptors. The IC50 value for competitive displacement of bound 125I-IGF-I was: 44 +/- 2 nM by IGF-I, 156 +/- 13 nM by IGF-II and 812 +/- 78 nM by insulin. The IC50 value for displacement of bound 125I-EGF was 0.37 +/- 0.03 nM by EGF and 0.42 +/- 0.04 nM by TGF alpha. In conclusion, only IGF-I and EGF receptors appear to be present on the apical surface of the pigmented rabbit bulbar conjunctiva. The IGF-I receptor is also capable of binding IGF-II and insulin, whereas the EGF receptor is also capable of binding TGF alpha.

Polycythaemia vera. IV. Specific binding of stem cell factor to normal and polycythaemia vera highly purified erythroid progenitor cells.

Polycythaemia vera (PV) patients' blood burst-forming units-erythroid (BFU-E) have an enhanced sensitivity to stem cell factor (SCF) compared to normal BFU-E. To characterize SCF receptors on erythroid progenitors from normal individuals and PV patients, we performed binding experiments using radioiodinated recombinant SCF (rSCF), day 1 BFU-E and day 8 erythroid colony-forming cells (ECFC), which are mostly colony-forming units-erythroid (CFU-E). 125I-rSCF binds to a single class of cell surface receptors (23,000/ECFC) at 0 degrees C with a high-binding affinity (Kd = 17 pM). Saturation occurred at 0.5 nM (10 ng/ml) which produces a nearly maximum biological effect. One half of the radiolabelled rSCF was internalized by the cells after 30 min at 37 degrees C. No significant differences in the receptor number, dissociation constant, or internalization rate were found between normal and PV ECFC. Autoradiographic analysis of 125I-rSCF binding to normal BFU-E and ECFC showed that no differences were present in either the percentage of positive cells or the number of radioactive grains/cell between the normal and PV erythroid progenitors. The enhanced sensitivity of PV BFU-E and CFU-E to SCF does not appear to be related to changes in SCF receptor number, binding affinity or internalization and the hypersensitivity of PV erythroid progenitors to SCF must reside in a further internal cellular abnormality.

Histomorphology, oLH and hCG receptors, and testosterone secretion in vitro in Rambouillet rams from lines in which females had been selected for low or high reproductive rate.

The objective of this study was to determine whether gross or histomorphological components of the testes, capacity and dissociation constants (Kd) of testicular oLH and hCG receptors, and gonadotrophin-stimulated testosterone secretion in vitro differed among Rambouillet rams from lines selected for low or high female reproductive rate and from rams of a random-bred control line. Lines had been selected for approximately 20 years. Data were collected from 22-month-old rams during the late breeding season. Rams among lines did not differ (P > 0.05) in gross testicular characteristics or most histomorphological characteristics. However, the percentage volume of interstitial vascular tissue was greater (P < 0.05) for rams from lines selected for low female reproductive rate than for rams from lines selected for high female reproductive rate. Receptor sites per Leydig cell and binding capacities of oLH and hCG receptors per testis, per gram of parenchyma, and per milligram of membrane protein did not differ (P > 0.05) among lines. The Kd values for oLH and hCG receptors did not differ (P > 0.05) among lines; however, receptor sites per Leydig cell, capacities of testicular parenchyma to bind gonadotrophin and Kd values were higher (P < 0.05) for oLH than for hCG receptors. Total oLH- and hCG-stimulated testosterone secretion in vitro did not differ (P < 0.05) among lines. In conclusion, selection for or against reproductive rate in Rambouillet ewes has not altered gross or most histomorphometric characteristics of the testes of male offspring, with the exception that selection against reproductive rate increased the proportion of testicular volume occupied by vascular tissue within the interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)

2,2′,4,4′,5,5′- and 3,3′,4,4′,5,5′-Hexachlorobiphenyl Alteration of Uterine Progesterone and Estrogen Receptors Coincides with Embryotoxicity in Mink (Mustela vison)

Female mink (Mustela vison) are highly sensitive to organochlorine (OC)-induced reproductive impairment. However, mechanisms of this reproductive toxicity are unknown. We have investigated the possible role of steroid receptors in embryotoxicity and reduced neonate weights. Anestrous, juvenile female mink and pregnant adult mink were exposed to 3,3′,4,4′,5,5′-hexachlorobiphenyl (3HCB), a coplanar polychlorinated biphenyl (PCB), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (2HCB), a noncoplanar PCB congener. Both congeners impaired 17 beta-estradiol-stimulated (24 hr after ip administration of 100 μg E2β ip) up-regulation of uterine nuclear estrogen receptors (ERn) in anestrous mink. Embryotoxicity and reduced embryo growth were first observed 14 days after exposure to 0.4 mg 3HCB/kg > 0.8 mg 3HCB/kg > 20 mg 2HCB/kg. In pregnant mink, all 3HCB treatments significantly increased progesterone receptor dissociation constants (PR Kd). ER concentration and PR total receptor number (Rt) were increased by 20 mg 2HCB/kg > 0.8 mg 3HCB/kg, but were unaffected by 0.4 mg 3HCB/kg. Serum E2β was below assay detection limits. Progesterone (P) concentrations were increased by 2HCB, decreased by 0.8 mg 3HCB/kg, and unchanged by 0,4 mg 3HCB/kg. Hepatic cytochrome P450 (P450) was induced 1.8-fold in anestrous and 2.2-foId in pregnant mink by 3HCB. Ethoxyresorufin-O-deethylase (EROD) was induced 13- and 4-fold in anestrous and pregnant mink, respectively. 2HCB exposure resulted in decreased P450 concentration in anestrous juveniles, but had no effect on P450 during gestation or EROD activity at any time. We propose that embryotoxicity and retarded embryo growth result from impairment of PR function and that differences in the efficacy of HCB treatments are a result of their dose-dependent, partial estrogenic actions which increase PR Rt via up-regulation of ER.

Differential expression of type I adrenal steroid receptors in immune tissues is associated with tissue-specific regulation of type II receptors by aldosterone.

We examined the influence of the type I adrenal steroid receptor agonist, aldosterone, on type II adrenal steroid receptor binding in the rat spleen and thymus after adrenalectomy. In the spleen, adrenalectomy was associated with a significant up-regulation of type II receptors, which was blocked by the concurrent administration of aldosterone (1 microgram/h) via sc osmotic minipumps. Neither adrenalectomy nor aldosterone treatment altered type II receptor binding in the thymus. Despite high doses of aldosterone (10 micrograms/h), which resulted in supra-physiological blood concentrations of this hormone, there was no evidence of type II receptor decreases in spleen or thymus below receptor levels found in sham-adrenalectomized rats. The effect of aldosterone on type II receptor binding appeared to be mediated by the type I receptor, since there was no aldosterone effect on the thymus, which did not exhibit detectable levels of type I receptor binding. Moreover, there was no evidence that aldosterone competed for the type II receptor in vivo or in vitro as determined by measurements of the type II receptor dissociation constant in the spleen of adrenalectomized, aldosterone-treated animals. Since selective activation of the type I receptor occurs in the spleen under physiological conditions, these results indicate that type I receptors may play a tonic inhibitory role in type II receptor expression in immune cells which express both receptor subtypes and reside in this tissue. Furthermore, the findings suggest that there may be different mechanisms involved in the up-regulation vs. the down-regulation of type II adrenal steroid receptors, and effects mediated solely via the type I adrenal steroid receptor appear only to influence the former process.

Differential expression of type I adrenal steroid receptors in immune tissues is associated with tissue-specific regulation of type II receptors by aldosterone

We examined the influence of the type I adrenal steroid receptor agonist, aldosterone, on type II adrenal steroid receptor binding in the rat spleen and thymus after adrenalectomy. In the spleen, adrenalectomy was associated with a significant up-regulation of type II receptors, which was blocked by the concurrent administration of aldosterone (1 microgram/h) via sc osmotic minipumps. Neither adrenalectomy nor aldosterone treatment altered type II receptor binding in the thymus. Despite high doses of aldosterone (10 micrograms/h), which resulted in supra-physiological blood concentrations of this hormone, there was no evidence of type II receptor decreases in spleen or thymus below receptor levels found in sham-adrenalectomized rats. The effect of aldosterone on type II receptor binding appeared to be mediated by the type I receptor, since there was no aldosterone effect on the thymus, which did not exhibit detectable levels of type I receptor binding. Moreover, there was no evidence that aldosterone competed for the type II receptor in vivo or in vitro as determined by measurements of the type II receptor dissociation constant in the spleen of adrenalectomized, aldosterone-treated animals. Since selective activation of the type I receptor occurs in the spleen under physiological conditions, these results indicate that type I receptors may play a tonic inhibitory role in type II receptor expression in immune cells which express both receptor subtypes and reside in this tissue. Furthermore, the findings suggest that there may be different mechanisms involved in the up-regulation vs. the down-regulation of type II adrenal steroid receptors, and effects mediated solely via the type I adrenal steroid receptor appear only to influence the former process.

Determination of steroid hormone receptors with the biochemical DCC method in endometrial cancer and tumor margin-endometrium relations to cellular changes and hormone level

In endometrial carcinoma and the surrounding marginal endometrium from 48 postmenopausal patients, the concentrations and dissociation constants of steroid hormone receptors were studied by the dextran-coated charcoal method (DCC), parallel to ploidy and morphology of tumour cell nuclei, grading, and the hormonal status. The results show, that the proportion of receptor-positive tumours is decreased in an advanced stage of disease. The mean receptor concentrations correlate both with nuclei parameters and the morphological results and reflect the process of dedifferentiation in the tumour centre. The higher receptor concentrations, especially the estradiol receptor concentration observed in the marginal endometrium, and the increased estradiol level in serum, demonstrate a raised tendency to proliferation in this region and could support the hypothesis, that a higher receptor level in adenomatous-hyperplastic tissues participates in tumour genesis. In patients with an advanced stage of the disease, determination of both receptors can yield indications for a purposeful adjuvant hormone therapy (anti-estrogen and/or gestagen), in addition to providing an aid to prognosis.

Effect of treatment with LHRH analogs containing cytotoxic radicals on the binding characteristics of receptors for luteinizing-hormone-releasing hormone in MXT mouse mammary carcinoma.

Binding capacities and apparent dissociation constants of receptors for luteinizing-hormone-releasing hormone (LHRH) were investigated in estrogen-independent MXT mammary cancers of untreated mice and after in vivo treatment with agonistic or antagonistic analogs of LHRH containing cytotoxic radicals: AJ-04 (agonist [D-Lys6]LHRH linked to methotrexate), T-98-([D-Lys6]LHRH coupled to glutaryl-2-(hydroxmethyl)anthraquinone (G-HMAQ)) and T-121/B (LHRH antagonist T-147 containing two residues of G-HMAQ), which induced tumor growth inhibition. The effects were compared to LHRH agonist [D-Trp6]LHRH and carriers [D-Lys6]LHRH, LHRH antagonist T-147, as well as to methotrexate, G-HMAQ and surgical bilateral overiectomy. Analysis of the binding data revealed that in control tumors the interaction of 125I-[D-TRP6]LHRH was consistent with the presence of one class of saturable, specific, noncooperative, high-affinity and low-capacity binding sites. Chronic treatment of mice bearing MXT tumors with LHRH analogs AJ-04 and T-121/B carrying cytotoxic radicals, but not with T-98 produced significant down-regulation of membrane receptors for LHRH. The largest decrease in dissociation binding constant and Bmax of receptors for LHRH was also found in animals treated with T-121/B. Specific, high affinity binding of 125I-labelled epidermal growth factor (EGF) was detected in the membranes from control and treated MXT tumors. Treatment with cytotoxic LHRH analogs, AJ-04, T-98 and especially with T-121/B, reduced maximal binding capacity of EGF receptors. Our results indicate that LHRH analogs carrying cytotoxic radicals retain their hormonal activity and inhibit tumor growth while inducing down-regulation of LHRH receptors. In addition, probably both components of the cytotoxic LHRH analog, peptide carriers and cytotoxic radicals, reduce the binding capacity of EGF receptors, which might be useful in the treatment of breast cancer.

Ovine maternal and fetal renal vasopressin receptor response to maternal dehydration

OBJECTIVE: Arginine vasopressin secretion increases in response to increased plasma osmolality or hypovolemia. Dehydration-induced increases in plasma arginine vasopressin levels have been shown to down-regulate arginine vasopressin V2 receptors in adult rat kidneys. Our study determined ovine maternal and fetal renal arginine vasopressin receptor characteristics and receptor response to maternal dehydration.STUDY DESIGN: Eight pregnant ewes (113 ± 1 days) were dehydrated for 72 hours; eight animals served as controls. Renal medullary tissue was isolated from maternal and fetal kidneys, and arginine vasopressin receptor characteristics determined with saturation and competition assays using tritiated arginine vasopressin, arginine vasopressin, and arginine vasopressin analogs.RESULTS: Euhydrated maternal and fetal renal medullary arginine vasopressin receptor dissociation constant (3.0 ± 0.3 and 1.9 ± 0.3 nmol/L) and maximal binding capacity (149 ± 15 and 111 ± 33 fmol/mg protein) values were similar. Pharmacologic profiles with selective agonists indicated a predominance of V2 receptors. Dehydration significantly increased maternal and fetal plasma osmolalities (304 ± 2 to 320 ± 2; 296 ± 1 to 319 ± 3 mOsm/kg water, respectively) and arginine vasopressin levels (3.8 ± 1.4 to 29.3 ± 4.6; 4.4 ± 1.0 to 16.9 ± 5.0 pg/ml, respectively) but had no effect on arginine vasopressin receptor binding.CONCLUSION: Specific, saturable, single-site tritiated arginine vasopressin binding is present in ovine maternal and fetal renal medullary membranes. Ovine maternal and fetal renal arginine vasopressin receptors do not down-regulate in response to dehydration-induced elevations in plasma arginine vasopressin levels. (AM J OBSTET GYNECOL 1992;167;1717-22.)

Myocardial sarcolemma isolated from skipjack tuna, Katsuwonus pelamis

The high cardiac outputs observed in tuna compared with other teleosts may imply differences in the regulation of myocardial contractility. Sarcolemma is a critical organelle in the teleost heart in the regulation of intracellular calcium concentration and hence contractility. A procedure is described for the isolation of large quantities of highly purified sarcolemma from the skipjack tuna heart by differential and sucrose-gradient centrifugation in the presence of multiple protease inhibitors. The preparation was characterized using the sarcolemma marker K+-stimulated p-nitrophenylphosphatase (K+pNPPase). From a starting ventricular wet weight of about 60 g, a yield of &gt; 20 mg sarcolemmal protein and a &gt; 43-fold purification over the crude homogenate were achieved. The percentage recovery of the total K+pNPPase in the sarcolemma fraction was greater than 14%. Both the purification index and recovery were substantially greater than those achieved with a much smaller scale sarcolemma preparation recently described for the trout heart. The equilibrium binding of the dihydropyridine PN200-110 and ryanodine were determined in these preparations. In purified sarcolemma, the dihydropyridine receptor binding density (Bmax) and dissociation constant (Kd) were found to be 0.48 ± 0.07 pmol∙mg protein−1 and 0.09 nM respectively, similar to that found in mammals. In crude homogenates, the ryanodine receptor Bmax was approximately 0.12 ± 0.04 pmol∙mg protein−1 and thus was a little more than half the value observed for the dihydropyridine receptor. The large-scale sarcolemma isolation procedure described in this paper for tuna may be useful for developing a better understanding of the role of the sarcolemma in the regulation of ventricular function in teleosts.

Altered peripheral benzodiazepine receptor density in human granulosa-lutein cells in relation to follicular maturity

Since the introduction of treatment by in vitro fertilization/ embryo transfer (IVF-ET) in humans, a harmless approach to identifying the ovum with a greater chance of producing an implantable embryo has been sought worldwide. Our previous studies indicated a high correlation between degree of follicular maturation and benzodiazepine (BZ) receptor density in rats. We hypothesized that if such a correlation existed in humans, the latter might be used to evaluate degree of follicular maturation and, consequently, its corresponding oocyte competence. We used [ 3H]PK 11195, a ligand selective to peripheral BZ receptors, to determine their density in human granulosa-lutein (G-L) cells. [ 3H]PK 11195 bound to G-L cells with high affinity in a saturable manner. Scatchard analysis revealed the presence of a single population of receptors. The average equilibrium dissociation constant and maximal number of receptors (B max) measured in G-L cells were 3.4 ± 0.2 nM and 608 ± 61 fmol/mg protein, respectively. In G-L cells obtained from individual follicles, a significant increase in the specific binding to peripheral BZ receptors was noted in G-L cells of follicles yielding oocytes at an advanced stage of cumulus maturation or oocytes competent for fertilization and subsequent cleavage. When G-L cells were pooled from all the follicles of each individual woman, the average B max value of [ 3H]PK 11195 in G-L cells was 900 ± 127 fmol/mg protein in women who conceived, which was 80% higher ( P < 0.01) than in women who did not conceive. We suggest that the remarkable increase in peripheral BZ receptor density in the G-L cells of individual follicles yielding fertilizable oocytes and in G-L cells of women who conceived should be considered as a fast and convenient predictor of the chance of pregnancy with human IVF-ET treatment.

Demonstration of Specific Binding of Cocaine to Human Spermatozoa

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.7 nmol/L) with or without unlabeled cocaine (670 mumol/L), and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23 degrees C. Competition studies with tritiated cocaine (3.4 to 66.6 nmol/L) indicated the presence of approximately 3.6 x 10(3) binding sites per cell, with a high affinity receptor dissociation constant (Kd = 12.6 nmol/L). Cocaine concentrations as high as 670 mumol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.

Progesterone receptor in the mammary tissue of pregnant and lactating gilts and the effect of tamoxifen treatment during late gestation

An isotope exchange assay using [3H]progesterone was used to examine progesterone receptor moieties in cytosolic extracts obtained from mammary tissue of gilts over the course of pregnancy and lactation, and during treatment of pregnant gilts with tamoxifen. Scatchard analysis was used to determine the concentrations and dissociation constants of progesterone receptors. The concentration of progesterone receptor was high at the onset of pregnancy (1394 fmol/mg DNA), fell to a nadir at 45 days (36 fmol/mg DNA), increased to a second maximum at 75 days (1232 fmol/mg DNA) and declined thereafter till parturition: the dissociation constant (Kd) of progesterone for its receptor remained stable during pregnancy with a mean Kd of 0.78 nmol/l. Progesterone receptors were not identifiable at day 21 of lactation. Treatment of pregnant gilts with tamoxifen (100 mg or 1.0 g/gilt per day orally identical to 0.70 or 7.0 mg/kg per day) did not affect the development of mammary structures or the ability to lactate at parturition; however, mammary progesterone receptor content in tamoxifen-treated animals tended to be lower than the controls at day 90 of pregnancy (15.7 +/- 1.56 vs 27.0 +/- 3.75 fmol/mg protein respectively). The results show that a temporal relationship exists between oestrogen concentrations in the circulation of pregnant gilts with progesterone receptor content in mammary tissue.

The labeling of lipoproteins for studies of cellular binding with a fluorescent lipophilic dye

N,N-dipentadecylaminostyrylpyridinium iodide is a dye that is approximately 100-fold more intensely fluorescent in a lipid than aqueous environment. This observation suggests its potential as a fluorescence stain for lipoproteins. This work reports the staining of LDL with this dye for use in studies of cellular binding. The staining procedure is simple, resulting in stable attachment of the dye as determined by transfer experiments, physical properties essentially identical to native LDL as demonstrated by virtually identical electrophoretic mobility, and consistent results in studies of cellular binding using flow cytometry. Increased signal to noise ratio over other dyes used for lipoprotein staining including the widely used Dil (3,3′-dioctadecylindocarbocyanine iodide) allows determinations of greater sensitivity and precision to be made. This is demonstrated by the flow cytometric determination of the 4°C binding curve of LDL with freshly isolated human peripheral blood lymphocytes (i.e., cells not LDL receptor upregulated). Mediation of binding by the LDL receptor is demonstrated by correspondence between the LDL receptor dissociation constant derived from this work and literature values; increased specific binding in lymphocytes cultured in lipoprotein-deficient media to up-regulate the LDL receptor; and decreased specific binding in lymphocytes cultured in the presence of 25-hydroxy cholesterol for 48 h to suppress the LDL receptor.