Discovery of a functionally selective ghrelin receptor (GHSR1a) ligand for modulating brain dopamine.

SignificanceThe modulation of growth hormone secretagogue receptor-1a (GHSR1a) signaling is a promising strategy for treating brain conditions of metabolism, aging, and addiction. GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable G protein- and ß-arrestin (ßarr)-dependent signaling pathways. Thus, pathway-selective modulation can enable improved pharmacotherapeutics that can promote therapeutic efficacy while mitigating side effects. Here, we describe the discovery of a brain-penetrant small molecule, N8279 (NCATS-SM8864), that biases GHSR1a conformations toward Gαq activation and reduces aberrant dopaminergic behavior in mice. N8279 represents a promising chemical scaffold to advance the development of better treatments for GHSR1a-related brain disorders involving the pathological dysregulation of dopamine.

From petting zoos to electronic classrooms: meeting the technology learning needs of family medicine teachers.

BACKGROUND AND OBJECTIVES: To meet the need for faculty development in the use of information technology for its membership, the Society of Teachers of Family Medicine (STFM) Program Committee implemented a pilot, fee-supported, electronic classroomformat at STFM's 2000 Annual Spring Conference. We assessed the characteristics of those who attended the sessions, the satisfaction of participants with the venue both from expressed satisfaction and enrollment, the financial viability of electronic classrooms, and whether participants used acquired skills 6 months after the conference. METHODS: An evaluation instrument was used to collect the demographic data on attendees and their satisfaction with the sessions they attended. This data was compiled and compared with the demographics of overall conference attendees. The enrollment and revenues for the electronic classrooms were totaled and compared with expenses. A 6-month post-conference phone survey was conducted to assess continued use of learned skills. RESULTS: Attendees were more likely to be physicians from community-based residencies. The program was filled to 80% capacity. Survey results indicated that the program was satisfying to attendees. Registration fees covered costs. Most participants were still using their new skills 6 months after the program. CONCLUSIONS: The electronic classroom pilot was successful and provides skills that participants use months after the program. This program can be used to meet the educational technology training needs of STFM members.

The effect of calcium-regulating hormones on transport of calcium across the chorioallantoic membrane of the chicken embryo.

The hormonal form of vitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), or appropriate vehicle were injected into the yolk sac of eggs of domestic fowl on days 16 and 17 of incubation. The chorioallantoic membrane (CAM) and overlying inner shell membrane were removed from eggs on day 18 and mounted in a Ussing-type apparatus. Transport of calcium was assessed by monitoring movements of radiolabeled calcium. Transport of calcium from the chorionic aspect of the CAM to the allantoic aspect increased considerably with time for all treatment groups except the one receiving PTH. "Back-flux" of calcium (movement of calcium from the allantoic aspect to the chorionic) was negligible for all treatment groups at all sampling periods. PTH treatment did not affect flux of calcium from allantois to chorion but reduced flux from chorion to allantois considerably. The underlying cause of this effect has not been identified. The hormonal form of vitamin D3 did not affect flux of calcium in either direction. These data raise the possibility that control of calcium transport by the CAM may not be the primary function of the vitamin D hormone.

Renal responses to parathyroid hormone in young chickens.

Renal clearance studies were performed in chicks 1, 5, and 9 days after hatching. Calcium gluconate was infused to block endogenous parathyroid hormone (PTH) secretion, whereas ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was infused to stimulate endogenous PTH secretion. PTH, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), or vehicle was administered intravenously. In 9-day-old birds, urinary cAMP and inorganic phosphate excretion fell dramatically after calcium loading and rose significantly after PTH, DBcAMP, or EGTA administration. These manipulations had no significant effect on excretion of calcium or inorganic phosphate in 1-day-old hatchlings. Five day-old-chicks gave an intermediate response. All three age groups, as well as 15-day-embryos, showed sharp increases in urinary cAMP values after PTH administration. Thus the adult response pattern to PTH appears to develop gradually during the first week after hatching or to be suppressed during the perinatal period, whereas the second-messenger response to the hormone, indicating hormone-receptor interaction, is present from late embryogenesis onward.

PTH-sensitive K(+)- and voltage-dependent Pi transport by chick renal brush-border membranes.

Brush-border membrane vesicles (BBMV) from chick (Gallus gallus) kidneys were used to examine possible pathways of Pi transport associated with Pi secretion. Preloading with 6 mM Pi trans-stimulated 32Pi uptake in the absence of Na+, indicating facilitation. Inside-positive voltage (100 mM K+, out > in, +valinomycin) increased Pi uptake from 161 +/- 4.4 to 241 +/- 16.1 pmol.mg protein-1.5s-1 at pH 7.5 (in = out). Gradients characterized by extravesicular pH (pHo) of 5.5 vs. intravesicular pH (pHi) of 7.5, 100 mM K+ (out > in), without and with valinomycin, further increased uptake to 664 +/- 148.5 and 946 +/- 90.8 pmol.mg protein-1.5s-1, respectively. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) had no effect on the latter response, but with 100 mM K+ (in = out), valinomycin decreased the response more than one-half, implicating a H+ diffusion potential. Generation of this potential with pHo 5.5 vs. pHi 7.5 and CCCP did not drive concentrative Pi uptake in absence of K+. Parathyroid hormone (PTH) treatment significantly increased this BBMV K(+)- and voltage-dependent Pi up-take compared with the parathyroidectomized (PTX) condition. The values of maximal uptake rate (Vmax) in PTH vs. PTX BBMV were 5,330 and 1,976 pmol.mg protein-1.5s-1, respectively. K(+)-dependent transport was inhibited by arsenate, phosphonoacetic acid, and vanadate. Together, the data indicate that this PTH-sensitive, voltage- and K(+)-dependent monovalent Pi transporter could be the mechanism by which Pi exits, cell-to-lumen, during renal tubular Pi secretion.

Ultrastructural localization of calcium in the chick yolk sac membrane endodermal cells as revealed by cytochemistry and X-ray microanalysis.

The yolk sac membrane (YSM) of the chick embryo transports calcium from the yolk into the embryonic circulation during the first half of development, but the intracellular pathway of calcium transport is poorly understood. In the present study, the ultrastructural localization of calcium was investigated in cells of the YSM of 9-day chick embryos. X-ray microanalysis as well as cytochemical techniques performed on yolk sac membrane cells treated with potassium oxalate, potassium ferricyanide and potassium antimonate demonstrated accumulation of calcium in yolk granules, digested yolk products, electron-dense bodies (EDBs; 100-400 nm diameter) and electron-dense granules (EDGs; 30-50 nm diameter). When strontium ions were injected into the yolk, they were incorporated into the endodermal cells and sequestered specifically in EDGs. From these results, we propose that calcium enters the endodermal cells by endocytosis of calcium-containing yolk granules, as well as through calcium channels in the apical cell membrane. In the cytoplasm, digested yolk products, EDBs, and EDGs act as sites of sequestration and accumulation of calcium. Extrusion of intracellular calcium into the extracellular space and embryonic circulation is accomplished by exocytosis of calcium-containing material and via an ion pump in the basal cell membrane.

Effects of cytochalasin B on calcium transport by 1,25(OH)2D3- or PTH-treated chick embryonic yolk sac in vitro and in vivo.

The present study was done to elucidate the mechanism(s) by which calcium is taken up or transported across the yolk sac membrane of the embryonic chick. We examined the effect of various inhibitors or experimental conditions on the uptake of calcium in vitro. Treatment with ouabain, verapamil, antimycin A and calcium ionophore A23187; substitution of choline for sodium or potassium in the buffer; or incubation of the tissue at 0 degree C had no significant effect on calcium uptake by the yolk sac membrane. Dinitrophenol (DNP) and lanthanum chloride (LaCl2) reduced 45Ca uptake from day 6 and 9 embryos by 15% and 30%, respectively. Cytochalasin B decreased the uptake of 45Ca in yolk sac membrane disks of day 6 embryos, but not in older embryos. The effects of cytochalasin B were explored further in embryos pretreated with either 1,25(OH)2D3 or PTH, both of which enhance calcium uptake. Cytochalasin B decreased calcium uptake in 9-day and 12-day vitamin D-treated embryos to about 60% of their hormone-enhanced level and also decreased PTH-stimulated 45Ca uptake into yolk sac disks by about 50% in embryos of all age groups tested. We also examined the effect of cytochalasin B on 45Ca transport across the yolk sac membrane in vivo. Cytochalasin B did not affect this transport in control (vehicle-treated) embryos. However, it significantly decreased the enhanced in vivo 45Ca transport in day-9 and -12 vitamin D-treated embryos approximately 30% and 45%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal clearance measurements of electrolytes in embryonic chickens.

Plasma ion concentrations and renal functions were examined in chick embryos after 9, 12, and 15 days of incubation. Plasma calcium and phosphate values rise steadily and significantly between days 9 and 15 of development. Values of other plasma ions (sodium, potassium, magnesium, sulfate) remain more or less the same during this developmental period. Glomerular filtration rates and urine flow rates decline steadily and significantly during this period of embryogenesis when expressed relative to body weight. Renal clearance values of calcium decrease between day 9 (16% of filtered calcium is excreted) and day 15 (4%). The latter value is the same as in birds after hatching and indicates that calcium is being conserved by the kidney of the chick during late embryogenesis. Other ions that appear to be conserved by the embryonic kidney are sodium and sulfate, whereas high percentages of filtered phosphate, potassium, and magnesium are excreted. Except for calcium, there was no significant difference between the renal handling of ions by the mesonephric kidney (functional in 9 day embryos) and the metanephric kidney (predominant in the 15 days embryos).

Ultrastructural localization of calcium in the chick chorioallantoic membrane as revealed by cytochemistry and X-ray microanalysis.

The chorioallantoic membrane (CAM) of the chick embryo actively transports calcium from the egg shell into the embryonic circulation. To investigate the intracellular pathway of calcium transport across the CAM, ultrastructural localization of intracellular calcium in cells of the chorionic ectoderm (CE) was determined using cytochemical methods and X-ray microanalysis. Treatment of the CE with potassium oxalate, potassium ferricyanide or potassium pyroantimonate revealed large numbers of electron-dense granules (EDGs) in the ectodermal cells. These measure 30-40 nm in diameter, and are not membrane-bound. These granules were seen in all three cell types of the CE. The presence of calcium in the EDG was directly confirmed by X-ray microanalysis. When strontium or barium ions were applied to the shell membrane side of the CAM, the cells of the CE incorporated these divalent cations and sequestered them in granules (25-40 nm in diameter) in cytoplasm and mitochondria. This study indicates that calcium enters the CE cells by means other than endocytosis, as the EDGs are not membrane-bound, that all three types of the CE cells appear to function in transport of calcium from shell to embryo during embryogenesis, and that the EDG plays important roles in intracellular accumulation of calcium during the process of calcium transport across the chorioallantoic membrane.

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks.

The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10(-4) M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na+ in the bathing solutions abolished the potential difference and the short-circuit current; when Na+ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 micrograms) and T3 (10 microM) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca2+ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na+ dependent and increases with developmental age in the chick embryo.

Compartmental analysis and glomerular filtration in chick embryos.

We have developed microtechniques that allow the determination of compartmental fluid distribution and glomerular filtration rate in chick embryos during three significant developmental periods: phase 1, the developmental period when the mesonephros alone is functioning; phase 2, periods of simultaneous meso/metanephric kidney function; and phase 3, the period during late development when the metanephros completely replaces the degenerated mesonephros. Water content of tissues is greater in younger embryos (89.4 +/- 0.2%, day 10) compared with older animals (78.3 +/- 0.5%, day 18). Although all major tissue components show an absolute increase in mass during this period, the embryo proper increases at five times the rate of the extraembryonic tissues. Glomerular filtration rate increases during development from 0.61 +/- 0.08 ml/h at day 10 to 2.31 +/- 0.11 ml/h at day 18. Glomerular filtration rate scales to body mass with an allometric exponent identical to adult birds only if total tissue mass (embryo + membranes) is considered. Our data suggest that significant errors in allometry will be encountered when scaling measurements are made on embryonic or fetal amniotes without taking into consideration the extraembryonic tissues.

Renal clearance of phosphate and calcium in vitamin D-deficient chicks: effect of calcium loading, parathyroidectomy, and parathyroid hormone administration.

Serum and renal clearance values of phosphate and calcium were measured and compared in 4 week-old vitamin D-deficient and vitamin D-replete chickens (Gallus gallus). D-deficient chicks had significantly lower body weights and serum calcium values; however, their renal functions were not different from D-replete controls. Serum calcium values in D-deficient birds did not change in response to parathyroid hormone (PTH) administration; however, they did drop significantly in response to parathyroidectomy (PTX). Serum phosphate values of D-deficient birds, but not D-replete birds, rose significantly after PTX. Clearance of phosphate is known to increase after administration of PTH. This conspicuous effect was absent in PTH-injected vitamin D-deficient chickens. PTX caused the excretion of phosphate to drop in both D-deficient and D-replete birds to near zero. Conversely, PTX of both D-deficient and D-replete chickens stimulated the excretion of more calcium than in controls. Calcium loading elevates the fractional excretion of calcium in both D-deficient and D-replete birds. It also causes a decrease in phosphate excretion in both groups, presumably by inhibiting the secretion of PTH. PTH administration to D-replete, calcium-loaded birds caused increased phosphate excretion (as it did in normal controls), an effect that was not seen in similarly treated D-deficient birds. Therefore, most renal functions studied after calcium loading, PTH administration, or PTX are not altered by vitamin D deficiency in the chicken. The major significant finding is that vitamin D-deficient chickens do not excrete increased amounts of phosphate in response to PTH stimulus.

Action of 1,25-dihydroxyvitamin D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos.

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, initially from the yolk and later from the eggshell. Calcium may be hormonally regulated during avian embryogenesis, but details of this regulation are lacking. We investigated the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], bovine parathyroid hormone [bPTH], and vehicle [ethanol or saline] on blood calcium values and incorporation of 45Ca into the yolk sac membrane of 9, 12, and 15 day chick embryos. Control data were also collected from uninjected 6 day embryos. Solutions were injected directly into the yolk sac compartment 48 and 24 hours prior to the experiment. Exogenous 1,25(OH)2D3 induced hypercalcemia in all age groups examined, while exogenous PTH induced hypercalcemia in day 12 and 15 embryos. Small disks of yolk sac membrane were incubated in medium to which 45Ca was added and assayed for 45Ca content at various intervals after start of incubation. In control yolk sac tissue, the uptake of 45Ca was greatest in younger embryos with decreasing uptake at developmentally more advanced ages; 1,25(OH)2D3 treatment significantly enhanced the uptake of 45Ca into yolk sac tissue in all groups (9, 12, and 15 day embryos). PTH treatment caused a significant elevation in 45Ca uptake in the day 12 and 15 embryos.

The avian embryo as a model for early developmental endocrinology.

Studies utilizing the developing chicken embryo have significantly augmented our understanding of the ontogeny of endocrine regulation during major critical periods of embryonic development. These embryos currently provide the only available models for elucidating the onset of endocrine function during all stages of in situ amniote development, for examining chronic calcium deficiency during embryogenesis, and for experiments in basic renal function during periods when only the mesonephros is normally functioning, as well as mixed meso/metanephric function and solely metanephric kidney function.

Glucocorticoid inhibition of Na-SO4 transport by chick renal brush-border membranes.

To examine the effect of glucocorticoids on sulfate transport by the chick (domestic Gallus gallus) renal tubule we dosed 3-wk-old animals with 60 micrograms dexamethasone/100 g body wt at 24 and 6 h before isolation of renal brush-border (BBM) and basolateral membranes (BLM). Dexamethasone treatment significantly reduced Na-dependent sulfate transport by BBM and had no effect in paired membranes on bicarbonate, proton, or electrical gradient-driven sulfate transport. The glucocorticoid treatment had no statistically significant effect on HCO3-SO4 exchange in the BLM. Kinetic analysis of the dexamethasone effect on the Na-SO4 transport process showed that apparent Vmax was significantly decreased to almost one-half that seen in controls (from 676 to 348 pmol.mg protein-1.5 s-1). The Km in control BBM was 0.40 +/- 0.095 mM and was not significantly different in dexamethasone-treated membranes (0.53 +/- 0.094 mM). To determine whether the dexamethasone-induced decrease in Na-SO4 transport by BBM was indirectly caused by stimulation of Na-H exchange and more rapid dissipation of the initial Na gradient used to drive sulfate uptake, we examined the effect of 0.1 mM amiloride on Na-SO4 uptake by BBM. With amiloride present, dexamethasone treatment caused Vmax to significantly drop from 1,102 to 660 pmol.mg protein-1.5 s-1. Amiloride had no statistically significant effect on the Km. The extent to which amiloride increased Na-SO4 transport and blocked 22Na uptake by BBM did not appear to be related to hormone treatment. The data indicate that glucocorticoids may participate in the regulation of sulfate excretion.

Ontogeny of vitamin D action on the morphology and calcium transport properties of the chick embryonic yolk sac.

The ontogeny of the calcium transport properties and hormonal modulation of the yolk sac membrane in amniote embryos is presently poorly understood. We investigated the role of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on plasma calcium values, yolk sac morphology and the ability of the yolk sac membrane to transport 45Ca from yolk to embryo. 1,25-(OH)2D3 treatment caused significant hypercalcaemia in 9-, 12- and 15-day embryos. Additionally, this hormone caused a hypertrophy of the endodermal cell layer that comprises the bulk of the yolk sac membrane. Both of these effects were the most dramatic in the 15-day embryo, the oldest age tested. 45Ca added to the yolk was transported into the blood rapidly across the yolk sac membrane. 1,25-(OH)2D3 significantly enhanced this transport in all age groups. [14C]Inulin was also taken across the yolk sac membrane, but at a slower rate than 45Ca; this transport was unaffected by 1,25-(OH)2D3. Thus, the yolk sac responds to 1,25-(OH)2D3 treatment both morphologically and functionally. The mechanism for transport appears to be a specific one, rather than a simple enhancement of non-specific endocytosis.

Sulfate transport by chick renal tubule brush-border and basolateral membranes.

Brush-border and basolateral membrane vesicles (BBMV and BLMV, respectively) were prepared from chick kidney by a calcium precipitation method and by centrifugation on an 8% Percoll self-generating gradient, respectively. In BBMV a 100-mM Na gluconate gradient, out greater than in, caused concentrative sulfate uptake approximately fivefold greater at 1 min than at 60 min (equilibrium) whether or not the membranes were short-circuited with 100 mM K gluconate, in = out, plus 20 micrograms valinomycin/mg protein. A 48-mM HCO3- gradient, in greater than out, stimulated a 2.5-fold higher uptake at 1 min than at 60 min, and short circuiting as above had no effect on the magnitude of this response. Imposition of a H+ gradient (pH 5.4 out vs. pH 7.4 in) caused concentrative uptake fourfold higher at 1 min than at equilibrium. Short circuiting as above or addition of 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited the pH gradient effect. Creation of an inside positive electrical potential with 100 mM K gluconate, out greater than in, plus valinomycin, also caused concentrative sulfate uptake. The K gradient in the absence of valinomycin had no effect on sulfate uptake (compared with isosmotic mannitol). Based on inhibitor/competitor effects, these are distinct sulfate transport processes. In chick BLMV, imposition of an HCO3- gradient, in greater than out, produced concentrative sulfate uptake; however, neither Na+ nor H+ gradients had significant effects at 15 s. 4-Acetamido-4'-isothiocyanostilbene 2,2'-disulfonic acid disodium at 0.1 mM was an effective inhibitor of BLMV bicarbonate-sulfate exchange; however, neither Cl-, SCN-, nor CCCP inhibited.

Calcium regulation in the embryonic chick. III. Calcium and phosphate in serum and allantoic fluid of normal and shell-less embryos.

Calcium metabolism of chicken embryos was profoundly affected by incubation in shell-less culture, but phosphate metabolism was largely undisturbed. Shell-less embryos exhibited hypocalcemia and hypocalciuria relative to normal embryos but had similar levels of phosphate in serum and allantoic fluid. The concentration of calcium in allantoic fluid declined during incubation in both groups, owing largely to accompanying increases in allantoic volume, but total amounts of calcium in the allantois did not vary with time. Both normal and shell-less embryos maintained higher concentrations of calcium in serum than in allantoic fluid, with shell-less embryos maintaining a larger gradient between serum and allantoic compartments. In contrast, serum and allantoic concentrations of inorganic phosphate increased over time in both normal and shell-less embryos, and both groups maintained generally higher concentrations of inorganic phosphate in the allantoic sac than in serum. Treatment of embryos with parathyroid hormone had no effect on calcium and phosphate metabolism. Embryos maintained in shell-less culture grew more slowly than those incubated normally and consequently had a dry mass about half that of normal embryos on day 18. Shell-less embryos also exhibited abnormalities in fluid balance, which were reflected in their inability to maintain normal allantoic volume and in their higher relative hydration compared to embryos incubated in ovo.

Effect of calcium supplementation on growth of shell-less cultured chick embryos.

Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth by 13 days of incubation (Dunn and Boone, Poult. Sci., 56:662-672, 1977). To determine whether addition of exogenous calcium might stimulate growth and/or survival of cultured embryos, calcium supplementation was attempted. Calcium supplementation between days 11 and 17 resulted in significant increases in both total embryo and serum calcium. Addition of shell pieces oriented with shell membranes onto the chorioallantoic membrane (CAM) resulted in significant stimulation of calcium transport by the CAM. However, growth of supplemented embryos was not increased to the same degree as were embryonic serum and total calcium levels. It is concluded that at least one factor other than calcium deficiency is responsible for retarded growth of shell-less cultured embryos.

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos.

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.