Ligand internalization and recycling by human recombinant somatostatin type 4 (h sst(4)) receptors expressed in CHO-K1 cells.

There is controversy as to whether somatostatin sst(4) receptors internalize. In this study, CHO-K1 cells expressing human sst(4) receptor (CHOsst(4) cells) cells internalized [(125)I]-[(11)Tyr]-SRIF in a time-dependent manner, reaching a steady state at 60 min (1.4+/-0.1x10(4) molecules internalized per cell). Internalization was blocked by hypertonic sucrose (0.5 M), ATP depletion or by decreasing the temperature to 4 degrees C. Internalization of [(125)I]-[(11)Tyr]-SRIF was also inhibited (pIC(50) values) by increasing concentrations of SRIF (7.74), L-362855 (6.27) and NNC-296100 (6.50) with pIC(50) values approximately 10 fold lower than those obtained for inhibition of [(125)I]-[(11)Tyr]-SRIF binding to membrane homogenates. Internalized ligand recycled rapidly to the extracellular media (t(1/2) 3.9+/-0.7 min) with only 6.8+/-0.6% of internalized radioactivity remaining in the cell after 45 min. Confocal microscopy of permeabilized, HA-epitope tagged CHOsst(4) cells labelled with a Cy-3 conjugated antibody revealed little internal immunostaining after SRIF (1 microM) treatment, consistent with the small proportion of receptors (3.5%) estimated to be internalized by radioimmunoassay. In summary, CHOsst(4) cells internalized [(125)I]-[(11)Tyr]-SRIF in a clathrin- and ATP-dependent manner with subsequent rapid recycling to the extracellular medium. Rapid receptor recycling and the consequent low proportion of receptors internalized at any one time may explain the inability to visualize internalized receptors by confocal microscopy. It seems unlikely therefore that the marked receptor desensitization observed in CHOsst(4) cells following SRIF treatment can be accounted for by a decrease in cell surface receptor expression.

Fates of endocytosed somatostatin sst2 receptors and associated agonists.

Somatostatin agonists are rapidly and efficiently internalized with the somatostatin sst2 receptor. The fate of internalized agonists and receptors is of critical importance because the rate of ligand recycling back to the cell surface can limit the amount of radioligand accumulated inside the cells, whereas receptor recycling might be of vital importance in providing the cell surface with dephosphorylated, resensitized receptors. Furthermore the accumulation of radioisotope-conjugated somatostatin agonists inside cancer cells resulting from receptor-mediated internalization has been used as a treatment for cancers that overexpress somatostatin receptors. In the present study, radio-iodinated agonists at the sst2 somatostatin receptor were employed to allow quantitative analysis of the fate of endocytosed agonist. After endocytosis, recycling back to the cell surface was the main pathway for both 125I-labelled somatostatin-14 (SRIF-14) and the more stable agonist 125I-labelled cyclo(N-Me-Ala-Tyr-d-Trp-Lys-Abu-Phe) (BIM-23027; Abu stands for aminobutyric acid), accounting for 75-85% of internalized ligand when re-endocytosis of radioligand was prevented. We have shown that there is a dynamic cycling of both somatostatin agonist ligands and receptors between the cell surface and internal compartments both during agonist treatment and after surface-bound agonist has been removed, unless steps are taken to prevent the re-activation of receptors by recycled agonist. Internalization leads to increased degradation of 125I-labelled SRIF-14 but not 125I-labelled BIM-23027. The concentration of recycled agonist accumulating in the extracellular medium was sufficient to re-activate the receptor, as measured both by the inhibition of forskolin-stimulated adenylate cyclase and the recovery of surface receptor number after internalization.

Evaluating the predictive validity of MCAT scores across diverse applicant groups.

PURPOSE: To examine the predictive validity of MCAT scores, alone and in combination with other preadmission data, for medical students grouped by race/ethnicity and sex. METHOD: This study included two samples: 1,109 students who entered in 1992 any of the 14 medical schools participating in the MCAT Predictive Validity Study; and all 11,279 students who entered medical school in 1992 and took the USMLE Step 1 in June 1994. Criterion measures included each student's cumulative GPA in the first two years of medical school and his or her pass/fail status on Step 1. Differential predictive validity was examined by comparing prediction errors across racial/ethnic and sex groups. For cumulative GPA; residuals were compared, and for Step 1, classification errors were studied. RESULTS: The patterns of prediction errors observed across the groups indicated that, on average, (1) no difference between the sexes in prediction errors was evident; (2) performances of the three racial/ethnic minority groups tended to be overpredicted, with significant findings for Asians and Hispanics; and (3) Caucasians' performance tended to be underpredicted, although the magnitude of this underprediction was quite small. When USMLE Step 1 scores were the criterion for success in medical school, the majority of errors were overprediction errors. CONCLUSION: The authors caution that although MCAT scores, alone and in combination with undergraduate GPA, are good predictors of medical school performance, they are not perfect. The authors encourage future research exploring additional predictor variables, such as diligence, motivation, communication skills, study habits, and other relevant characteristics. Similarly, they indicate that high grades and Step 1 scores are not the only indicators of success in the medical profession and call for studies examining other important qualities, such as integrity, interpersonal skills, capacity for caring, willingness to commit to lifelong learning, and desire to serve in underserved areas.

The relationship between agonist intrinsic activity and the rate of endocytosis of muscarinic receptors in a human neuroblastoma cell line.

The molecular mechanisms underlying the internalization of G protein-coupled receptors are still poorly understood. Normally agonists but not antagonists cause internalization (defined here as a reduction in the number of receptors at the cell surface), suggesting a functional relationship between agonist activity and internalization. In this study we investigated the effects of eight muscarinic ligands on the rate constants for endocytosis and recycling of m3 muscarinic acetylcholine receptors in human SH-SY5Y neuroblastoma cells. We found that there was a linear correlation between the intrinsic activity of the ligand and its ability to increase the rate constant for endocytosis, suggesting that the same active conformation of the receptor is responsible for stimulating both second messenger generation and receptor endocytosis. In contrast, the rate constant for recycling did not depend on which agonist had triggered receptor endocytosis, suggesting that recycling is a purely constitutive process. Because receptor internalization depends on the rate constants for both endocytosis and recycling, the relationship between internalization and intrinsic activity is nonlinear. In particular, mathematical modeling of receptor trafficking revealed that under certain conditions very small (3% or less) increases in the rate constant for endocytosis are sufficient to cause substantial receptor internalization. An important implication of this analysis is that extremely weak partial agonists (which may in practice be indistinguishable from antagonists) may produce significant receptor internalization.

Involvement of receptor cycling and receptor reserve in resensitization of muscarinic responses in SH-SY5Y human neuroblastoma cells.

Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca2+ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca2+ response to 1 mM carbachol is approximately 50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.

Endocytosis and recycling of G protein-coupled receptors.

Agonist stimulation of G protein-coupled receptors causes a dramatic reorganization of their intracellular distribution. Activation of receptors triggers receptor endocytosis and, since receptors recycle back to the surface continuously, a new steady state is reached where a significant proportion of receptors is located internally. Although this movement of receptors is remarkable, its role has been enigmatic. Recent developments have provided insight into the compartments through which the receptors move, the nature of the signals that trigger receptor translocation, and the significance of receptor cycling for cell function. In this article, Jennifer Koenig and Michael Edwardson review recent progress in this field and place receptor cycling into a mathematical framework that reveals the extent and rate of intracellular receptor movement.

Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics.

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF1-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [125I]-Tyr11-SRIF-14, with a pIC50 of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF1 receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst2 subtype. 2. The stable radioligand, [125I]-BIM-23027, bound with high affinity (Kd = 13 pM, Bmax = 0.2 pmol mg-1 protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4 degrees C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C and 89% of the ligand dissociated at 37 degrees C. 3. Equilibrium binding of [125I]-BIM-23027 was partially (25%) inhibited by 10 microM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [125I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 microM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 degrees C. Binding was totally inhibited by pretreatment of cells with pertussis toxin. 4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC50 of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst2 receptor, no rise in intracellular calcium concentration was observed with SRIF-14. 5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst2 type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization.

1. Receptor-dependent internalization of somatostatin (SRIF) agonists has been a matter of controversy probably because [125I]Tyr11-SRIF-14 is rapidly degraded. We have studied the internalization of a stable somatostatin analogue, [125I]-BIM-23027, in a neuronal cell line, Neuro2A, which natively expresses somatostatin sst2 receptors. 2. Incubation of Neuro2A cells with [125I]-BIM-23027 at 37 degrees C resulted in a time-dependent internalization of the ligand, which reached a maximum at 30 min. Acid-washing showed that cell-surface binding of the ligand accounted for only 34% of total binding at this time. Internalization was dramatically reduced at 15 degrees C. 3. Internalization of [125I]-BIM-23027 was prevented by inclusion of unlabelled somatostatin receptor agonists in a concentration-dependent manner. The IC50 values for inhibition of [125I]-BIM-23027 internalization were approximately 100 fold lower than for inhibition of [125I]-BIM-23027 binding to membrane homogenates but followed the same rank order of potencies. 4. Disruption of G-protein coupling by treatment with pertussis toxin caused a 60% reduction in internalization of ligand. A combination of antimycin (50 nM) and deoxyglucose (50 mM) pretreatment, which leads to a depletion of cellular ATP, decreased internalization of [125I]-BIM-23027 by 66% of control and increased the proportion of surface-bound ligand. Hypertonic sucrose, which prevents clathrin-mediated endocytosis, reversibly abolished the internalization of ligand without increasing the proportion bound at the cell surface. 5. After internalization of [125I]-BIM-23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37 degrees C. This finding suggests that the intracellular content of [125I]-BIM-23027 reaches a steady state which is determined by the rates of both internalization and recycling of the ligand. In contrast to studies in which the internalization of [125I]-Tyr11-SRIF-14 was examined, neither internalized nor recycled [125I]-BIM-23027 was degraded to its component amino acids. 6. These findings indicate that the somatostatin agonist, [125I]-BIM-23027, is internalized in a receptor-dependent manner which involves clathrin-coated pits in Neuro2A cells. Furthermore, much of the internalized ligand is rapidly recycled back to the extracellular medium without undergoing significant degradation.

Intracellular trafficking of the muscarinic acetylcholine receptor: importance of subtype and cell type.

Agonist-induced decrease of surface muscarinic receptor number occurs in a number of cell lines. Recent work has suggested that some muscarinic receptor subtypes undergo internalization, whereas others do not. We investigated the agonist-induced trafficking of various muscarinic receptor subtypes transfected into CHO cells and compared it with the trafficking of receptors expressed natively in neuronal cells, fibroblasts, or epithelial cells. SH-SY5Y neuroblastoma cells, which express predominantly the m3 receptor subtype, show qualitatively similar changes in surface receptor number in response to agonist stimulation to those occurring in NG108-15 cells, which express predominantly the m4 subtype. The rate constants for internalization, however, were considerably different, indicating that receptors in SH-SY5Y cells show a much faster turnover than those in NG108-15 cells. In the transfected cells, the muscarinic receptor subtypes m1 and m3, which are coupled to second messenger systems via Gq/11, showed little agonist-induced loss of surface receptors. In contrast, the muscarinic receptor subtypes m2 and m4, which are coupled via Gi or G(o), showed a substantial loss of surface receptors after treatment with agonist. An interesting implication of this result is that agonist-induced receptor trafficking can still occur efficiently, even at very high receptor densities. Significant agonist-induced internalization also occurs in a fibroblast line (HeLa) and an epithelial cell line (HT29), both of which express predominantly m3 receptors. Our results suggest that the extent and rate of the loss of receptors from the cell surface in response to agonist stimulation are governed by both the receptor subtype and the cell type in which it is expressed.

Kinetic analysis of the trafficking of muscarinic acetylcholine receptors between the plasma membrane and intracellular compartments.

We have studied the kinetics of muscarinic receptor trafficking between the plasma membrane and intracellular compartments in NG108-15 cells. A model of trafficking is proposed that includes four transport steps, delivery to the plasma membrane from the Golgi complex (linear) and endosomes (exponential) internalization from the plasma membrane into endosomes (exponential), and a degradative route from endosomes into lysosomes (linear). Based upon this model, a general equation has been derived that describes changes in receptor number at the plasma membrane and in endosomes in response to agonist stimulation. By following the movement of receptors into and out of the plasma membrane under various experimental conditions, it has been possible to determine the values of the four rate constants in the general equation and also the size of the endosomal receptor pool created by agonist stimulation. The consequences of changes in these constants for receptor trafficking are demonstrated. The model accounts for the effect of varying the duration of agonist stimulation on the size of the endosomal receptor pool and also permits an estimation of the receptor trafficking that underlies the well-established phenomenon of agonist-induced receptor internalization.

Routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in NG108-15 cells.

1. We have examined the routes of delivery of muscarinic acetylcholine receptors to the plasma membrane in unstimulated and agonist-stimulated NG108-15 cells. Delivery of receptors to the plasma membrane was measured by irreversible alkylating receptors already at the cell surface with propylbenzilylcholine mustard (PrBCM) and then following the recovery of binding of the polar radioligand [3H]-N-methylscopolamine ([3H]-NMS) in intact cells. 2. In unstimulated cells, recovery of [3H]-NMS binding after 2 h of incubation at 37 degrees C was 30% of binding in control cells. Binding affinity of [3H]-NMS was unchanged. In cells that had been pre-exposed to carbachol (0.5 mM) for 30 min, initial [3H]-NMS binding was reduced by 38% but recovery of binding was increased from 30% to 43% of control binding. 3. When the cells were pre-incubated for 1 h with the protein synthesis inhibitor cycloheximide (20 micrograms ml-1), recovery of [3H]-NMS binding was reduced by similar extents in unstimulated (30% to 9%) and carbachol-stimulated (43% to 19%) cells. Incubation of the cells at 20 degrees C instead of 37 degrees C reduced recovery of [3H]-NMS binding from 30% to 9% in unstimulated cells and from 43% to 23% in carbachol-stimulated cells. 4. Depletion of cellular ATP by addition of antimycin (50 nM) and deoxyglucose (50 mM), reduced recovery of binding to 12% in unstimulated cells and to 6% in carbachol-stimulated cells. 5. These results indicate that in unstimulated NG108-15 cells, delivery of muscarinic receptors to the plasma membrane is almost exclusively through the synthetic pathway. In agonist-stimulated cells,receptor sequestration into an intracellular compartment (probably endosomes) occurs. Under these circumstances, delivery of receptors to the plasma membrane along the synthetic route is unaffected but an additional route of delivery (recycling) now operates.

Effect of free fatty acids on GABAA receptor ligand binding.

Phospholipase A2 (PLA2) treatment of synaptosomal membranes, which causes the release of fatty acids, particularly unsaturated fatty acids, inhibits the flux of chloride ions through the gamma-aminobutyric acid (GABA) benzodiazepine receptor ion channel in response to activation by agonists. PLA2 treatment has also been shown to affect ligand binding to the receptor. In the present study, we have investigated the effect of unsaturated free fatty acids, arachidonic acid and oleic acid and saturated free fatty acids, arachidic acid and stearic acid on various characteristics of GABAA receptor ligand binding. Only the unsaturated fatty acids showed any effect: arachidonic acid and oleic acid enhanced flunitrazepam binding and muscimol binding but inhibited tert-butylbicyclophosphorothionate (TBPS) binding in a dose-dependent manner. The effects on muscimol and TBPS binding were shown to be due to changes in receptor density by saturation analysis. Oleic acid and arachidonic acid also decreased the enhancement of flunitrazepam and muscimol binding by cartazolate and pentobarbital but did not affect GABA enhancement of flunitrazepam binding. These data indicate that unsaturated free fatty acids can mimic the effects of PLA2 treatment and underline the importance of the lipid microenvironment on ligand binding to the GABAA receptor.

Comparison of medical school performances and career plans of students with broad and with science-focused premedical preparation.

This paper reports (1) a method for classifying students according to the breadth of their premedical preparation and (2) a comparison of the medical school performances and career plans of the students thus classified. The method was developed in 1987, in part by using input from a small but representative sample of admission officers. Students were grouped according to undergraduate major, ratio of nonscience-to-science course hours, and extracurricular involvement. After tentatively classifying all individuals who had entered U.S. medical schools in 1981 as having either broad or science-focused preparation, the author compared the two most distinct groups selected from a random sample of the individuals in each classification: 59 individuals constituted the final broadly prepared group, and 73, the science-focused group. The science-focused group attained higher mean scores (p less than .05) on three science sections of the National Board of Medical Examiners (NBME) Part I examination, and the broadly prepared group scored higher on the Behavioral Sciences section (p less than .05). No other significant difference was evident between the groups' mean scores on the NBME Parts I, II, or III, or in the groups' rates of experiences of academic difficulty, specialty choice distributions, or percentages of individuals deciding to pursue research careers. The author concludes that this method of classifying students is useful and that the students with less premedical focus in the sciences were able to perform well.

Works in progress #2. Threaded acetabular components. Design rationale and preliminary clinical experience.

Two design rationales for threaded acetabular components, the S-ROM Anderson cup and S-ROM SuperCup, are discussed. Components have been followed for at least six months, with a range of six to 24 months. The average follow up is nine months. One hundred components in 96 patients had sufficient follow up and full clinical and roentgenographic evaluations to be included in this study. The 100 hips were divided into four groups based on S-ROM Anderson cup versus S-ROM SuperCup and on primary versus revision. Each hip was evaluated on a clinical and radiologic basis. Based on clinical and roentgenographic evaluations of both the primary and revision situations of the S-ROM SuperCup, consistently good to excellent results were observed. To date, no case has undergone revision for clinical or roentgenographic failure. Dramatic pain relief was exhibited by 96% of patients. Although early results are very encouraging, longer follow ups are necessary.

An interim report on the MCAT Essay Pilot Project.

Results from four pilot administrations of the Medical College Admission Test essay question are reported. Analyses focused on (a) the performance characteristics of sample groups differentiated by gender, size of hometown, race/ethnicity, and dominant language; (b) the relationships between essay scores and academic/demographic characteristics; and (c) the reliability of one 45-minute versus two 30-minute essays. No differences were found for examinees grouped by gender and size of home community. Mean differences among the racial/ethnic groups were explained largely by reading level differences. Differences in essay performance by language group were large and unexplained by reading level differences. No relationship was found between the essay score and the academic/demographic characteristics. Reliability estimates for two 30-minute essays were higher than for one 45-minute essay; however, the 30-minute period yielded writing of poorer quality. Test-retest reliabilities for the 45-minute topics will remain the focus of future studies as will performance by examinees for whom English is a second language. The impact of the essay on the selection process will also be assessed.

Flavin-containing monooxygenase activity in human liver microsomes.

Human liver microsomal flavin-containing monooxygenase activity has been studied using dimethylaniline N-oxidation and thiobenzamide S-oxidation. Except for one subject, the capacity of human liver microsomes to mediate these reactions were markedly increased at pH 8.4 compared to pH 7.4. The mean dimethylaniline N-oxidase activities at pH 7.4 and 8.4 in the four subjects tested were 2.49 +/- 1.13 and 6.59 +/- 4.04 nmol mg-1 min-1, respectively (mean +/- SD, N = 4). The mean thiobenzamide S-oxidase activities at pH 7.4 and 8.4 were 1.39 +/- 0.51 and 2.74 +/- 1.28 nmol mg-1 min-1, respectively. At pH 7.4, an antibody to the human liver NADPH-cytochrome P-450 reductase inhibited dimethylaniline N-oxidation between 4 and 38%. The same antibody had no effect on this reaction at pH 8.4. Except for one subject, a battery of cytochrome P-450 inhibitors also had little effect on this reaction. Further, preincubating human microsomes at 45 degrees C in the absence of NADPH for 4 min destroyed approximately 90% of the dimethylaniline N-oxidase activity. These data collectively suggested that the flavin-containing mono-oxygenase is the major enzyme mediating this reaction in human liver microsomes. In contrast to dimethylaniline N-oxidation, thiobenzamide S-oxidation was significantly inhibited by the anti-reductase at both pH 7.4 and 8.4, respectively. These data indicate that cytochromes P-450 contribute significantly to this reaction in human liver microsomes.