Antitumor effects of analogs of LH-RH and somatostatin: Experimental and clinical studies

Many clinical approaches for the treatment of hormone-sensitive tumors are being developed based on analogs of LH-RH and somatostatin. Inhibition of the pituitary-gonadal axis forms the basis for oncological applications of LH-RH agonists like [ -Trp⁶]-LH-RH and new LH-RH antagonists free of edematogenic effects such as [Ac- -Nal(2)¹- -Phe(4Cl)²- -Pal(3)³, -Cit⁶, -Ala¹⁰]-LH-RH (SB-75). Agonists and antagonists of LH-RH have been used in patients with prostate cancer and might be also beneficial for the treatment of breast cancer and ovarian, endometrial and pancreatic carcinomas. Some of the effects of LH-RH analogs can be due to direct action since LH-RH receptors have been found in these cancers. The use of sustained delivery systems based on microcapsules of PLG, makes the treatment more efficacious. Octaeptide analogs of somatostatin such as -P s-Trp-NH₂ (RC-160) and related analogs were designed specifically for antitumor activity. These somatostatin analogs, by virtue of having a wide spectrum of activities appear to inhibit various tumors through multiple mechanisms. Direct antiproliferative actions of somatostatin analogs appear to be mediated by specific receptors located on tumor cells. High affinity binding sites for RC-160 and related analogs have been found in human pancreatic, prostate, breast and ovarian cancers and brain tumors such as meningiomas. In vivo administration of analog RC-160 inhibits the growth of Dunning R-3327 prostate cancers in rats, MXT mammary tumors in mice and BOP-induced ductal pancreatic cancers in hamsters. Combination of microcapsules of RC-160 with [ -Trp⁶]-LH-RH results in synergistic potentiation of the inhibition of these cancers. Somatostatin analog RC-160 and LH-RH antagonist SB-75 are the object of further experimental studies and clinical trials aimed at the exploration of their inhibitory effects on the processes of malignant growth.     

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.     

2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

The binding of bombesin and somatostatin and their analogs to human colon cancers.

Specific receptors for bombesin/gastrin-releasing peptide, somatostatin, and EGF were investigated in 15 human colon cancer specimens. Eight of 15 clinical specimens (15%) of colon cancer showed the presence of somatostatin receptors. Octapeptide somatostatin analogs, RC-160 and RC-121, showed 10 times higher binding affinity for somatostatin receptors on colon cancer membranes than somatostatin. Analysis of 125I-Tyr4-bombesin binding data revealed the presence of specific binding sites in six (40%) specimens of human colon cancer. Scatchard analysis of 125I-labeled bombesin indicated a single class of receptors in three specimens with an apparent Kd value of 2.5 nM and two classes of receptors with high (Kd = 0.4 +/- 0.2 nM) and low affinity (Kd = 1.6 +/- 0.4 microM) in three other specimens. The 125I-Tyr4-bombesin binding capacities in the colon cancers for high affinity binding sites were from 6 to 228 fmol/mg protein and for low affinity binding sites 76 +/- 15 pmol/mg protein. None of the membrane preparations made from normal colonic mucosa specimens showed specific binding for 125I-Tyr4-bombesin. Five pseudononapeptide (psi 13-14) bombesin (6-14) antagonists, with different modifications at Positions 6 and 14, synthesized in our laboratory, inhibited the binding of 125I-Tyr4-bombesin in nanomolar concentrations. No correlation was found between the degree of differentiation and the presence of binding sites for somatostatin or bombesin. Specific binding of EGF was detected in 80% of colon cancer specimens. EGF binding capacity in colon cancer membranes was on average twice as high as in normal colon mucosa (50 +/- 21 vs 28 +/- 14 fmol/mg protein, respectively). Specific binding sites for somatostatin and EGF, but not bombesin, were also demonstrated in human colon cancer cell line HT-29. In HCT-116 colon cancer line only EGF receptors were found. These receptor findings and our in vivo studies on inhibition of colon cancer growth support the merit of continued evaluation of somatostatin analogs and bombesin/gastrin-releasing peptide antagonists in the management of colonic carcinoma.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of specific vasopressin binding sites in cell membranes of the adenohypophysis, liver, kidney and adrenal glands of the rat

3H vasopressin specifically binds to the binding sites in liver, kidney and adenohypophysis with Bmax = 11.8 +/- 5.6, 1.7 +/- 0.5 and 4.9 +/- 1.2 pmol/g tissue and Kd = 1.5 +/- 0.5, 0.66 +/- 0.21 and 0.84 +/- 0.21 nM, correspondingly. Specific binding increases in the presence of Mg2+ and Ni2+ and decreases at high temperature (37 degrees). The presence of high affinity binding sites for 3H vasopressin was shown in the rat adrenals, binding was saturable and reversible. Concentration of vasopressin binding sites in adrenals is 6-8 fold less than in adenohypophysis. It is supposed that vasopressin receptors in adrenals may participate in the regulation of corticosteroid secretion.     

Interaction of alpha-(125)latrocrustotoxin with nerve cell membranes from the river crab Astacus astacus]

alpha-Latrocrustatoxin, the crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus was radioactively labelled with Bolton-Hunter reagent to the specific activity of 160 Ci/mmol with retention of the biological activity. A highly specific binding of radioactive toxin on plasmatic membranes from the crayfish Astacus astacus nerve cells with Bmax = 0.04 pmol binding toxin/mg membrane protein and Kd = 0.7 x 10(-10) M was demonstrated.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of gastric mucosal prostaglandin E2 receptor

1. The binding characteristics of gastric mucosal prostaglandin (PG) E2 (PGE2) receptor were investigated using mucosal cell membranes from rat stomach. The binding was found to be dependent upon PGE2 and membrane protein concentration, the time of incubation and the pH of the mixture, being highest at pH 3.0. 2. Scatchard analysis of the binding data revealed a curvilinear plot with high affinity binding (Kd = 2 nM; Bmax = 0.106 pmol/mg protein) and low affinity binding (Kd = 319 nM; Bmax = 2.262 pmol/mg protein) sites. 3. Competitive displacement study indicated that the receptor was specific for PGs of the E series, as PGF2 alpha and 6-keto-PGF1 alpha failed to displace the PGE2. 4. The study is the first report to provide biochemical parameters of specific PGE receptors in rat gastric mucosa.     

Glucose transporters in chromaffin cells: subcellular distribution and characterization

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Administration of chlordiazepoxide affects [3H][3-MeHis2]thyrotropin-releasing hormone binding in rat brain.

The effects of acute and subchronic administration of chlordiazepoxide (CDZ) on [3H][3-methyl-histidyl2]thyrotropin-releasing hormone binding to thyrotropin-releasing hormone (TRH) receptors in membrane preparations from various regions of rat brain were examined. Acute administration of CDZ (50 mg/kg x 3 within 24 h) did not alter either the equilibrium dissociation constant (Kd) or the maximum number of binding sites (Bmax) in cerebellum (CB), olfactory bulbs (OB), frontal cortex (Cx), hypothalamus (HT) or corpus striatum (ST). However, the Kds of the pyriform cortex/amygdala (PC/A) (Kd = 3.6 +/- 0.1 nM compared to 1.9 +/- 0.1 nM in the control group; p less than 0.01) and the hippocampus (HP) (Kd = 7.8 +/- 0.7 nM compared to 2.1 +/- 0.1 nM in the control group; p less than 0.01) were increased. There were no changes in Bmax. Subchronic administration of CDZ (50 mg/kg/day for 7 days) increased the Kd of the PC/A complex (p less than 0.05), the OB (p less than 0.05) and the HP (p less than 0.01) without altering in Bmax. These results, showing regional differences in the response of TRH receptors to acute and subchronic CDZ administration, suggest that reduced affinity of TRH receptors in the PC/A complex, OB and HP may be related to some of the neurobiological actions of CDZ and/or its metabolites.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.     

1,4-Dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts

By using the radioactively labeled 1,4-dihydropyridine (DHP) probe, [3H]PMD, we have demonstrated that cultured human embryonic fibroblasts grown at a low density in Eagle's medium supplemented with serum contain a single class of non-interacting DHP binding sites (Bmax, 1.2 +/- 0.3 pmol/10(6) cells; Kd, 3.9 nM). After inhibition of the DHP receptor biosynthesis by cycloheximide, the number of [3H]PMD binding sites is reduced with a half-time of 12 h, which implies a turnover rate of 30,000 +/- 7500 receptors/h per cell. With progression to confluency, the Bmax value decreased up to 0.28 +/- 0.08 pmol/10(6) cells without significant change in Kd value. When cells were grown at a low density in serum-free conditions, the number of [3H]PMD binding sites gradually increased 1.9-fold within 3 days. Addition of serum reversed this effect with the same time course. These results imply that the DHP-sensitive Ca2+ channels are involved in the control of the proliferation of human embryonic fibroblasts.     

Characterization of the epidermal growth factor receptor in preimplantation pig conceptuses.

Embryos recovered from sows on Days 9-13 of pregnancy (Day 0 = first day of estrus) exhibited saturable and time-dependent specific binding of 125I-epidermal growth factor (EGF). The specific binding (pg/mg protein) was greater (P less than 0.001) for Day 13 elongated conceptuses than for conceptuses of earlier stages. Scatchard analyses showed two classes of binding sites (Kd = 7.0 +/- 2.6 x 10(-11) M, Bmax = 6.2 +/- 1.4 fmol/mg protein and Kd = 3.4 +/- 0.2 x 10(-8) M, Bmax = 420 +/- 80 fmol/mg protein). The EGF receptor in Day 13 conceptus membranes is a 170-kDa protein and was phosphorylated in the presence of EGF and adenosine triphosphate. EGF stimulated protein tyrosine kinase activity about 1.6-fold over basal levels. The results show that the preimplantation pig conceptus possesses EGF-binding sites with the properties of functional EGF-receptors.