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.

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.

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.

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.

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.

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.

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 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.

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.

Avian medullary bone in organ culture: effects of vitamin D metabolites on collagen synthesis.

A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of 3H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10-15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) resulted in a dose-dependent inhibition of 3H-proline incorporation into CDP at doses from 10(-10)M to 10(-7)M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since 3H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)2D3-induced decrease in the 3H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)2D3 is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaired incorporation of collagen into bone matrix, or bone resorption. Medullary bone collagen synthesis was not affected by 24,25(OH)2D3, either alone or in combination with 1,25(OH)2D3. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)2D3 and 1,24,25(OH)3D3 were the most potent metabolites tested, followed by 1-alpha(OH)D3. 25(OH)D3 and 24,25(OH)2D3 had no effect at concentrations as high as 10(-7)M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as an in vitro model for the study of bone formation.

Renal excretion in gull chicks: effect of parathyroid hormone and calcium loading.

Renal clearance experiments were performed on herring gull (Larus argentatus) and great black-backed gull chicks (L. marinus) to test the importance of parathyroid hormone (PTH), parathyroidectomy (PTX), and calcium loading on excretion patterns of sodium, potassium, calcium, and phosphate. PTX reduced the relative clearance of phosphate to near zero within 2 h. Conversely, PTH stimulated net secretion of phosphate within 40 min. Calcium loading caused a drop in phosphate excretion identical to that of PTX, presumably by inhibiting PTH secretion by the parathyroid glands. This also could be reversed by administration of PTH. Serum phosphate values were highest in PTX gulls and lowest in calcium-loaded gulls. The relative clearance of calcium (CCa/CIn) was elevated in calcium-loaded birds. However, CCa/CIn did not change significantly in response to either PTX or PTH in the sham-operated or PTX birds. Serum calcium values were highest in calcium-loaded gulls and lowest in PTX gulls, the reciprocal of the effects on serum phosphate.

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.

Release of calcium from bone in vitro by homogenates of embryonic chick parathyroid glands.

Parathyroid glands were removed from chick embryos of 12 to 20 days of development and tested for their ability to stimulate calcium release from calcium-45-labeled embryonic chick bone in organ culture. All glands stimulated calcium-45 release to approximately the same degree, although the glands of 16- and 20-day embryos had a somewhat enhanced calcium-mobilizing ability. It appears that parathyroid hormone is present in the embryonic chick coincidental with the time of early development of the skeleton and is present in similar quantities throughout the remainder of embryogenesis.

Calcitonin stimulation of urine flow and sodium excretion in the starling.

Renal effects of synthetic salmon calcitonin (CT) were examined in normal and parathyroidectomized (PTX) starlings, Sturnus vulgaris. Standard clearance studies were performed. Anesthetized birds were infused with [14C]-inulin in 2.5% mannitol containing either 0.25% gelatin alone as a control, or gelatin plus CT (2 IU CT/h; Armour). In intact starlings, CT significantly increased urine flow and relative clearance of sodium (CNa/CIn). CCa/CIn was significantly elevated only 40 min after start of hormone infusion. No significant change occurred in CK/CIn, CPO4/CIn or in serum levels of Na, K, Ca, or PO4. In PTX starlings, CT induced significantly higher CNa/CIn. Urine flow was significantly higher at 40 and 60 min, whereas CK/CIn increased significantly only 20 min following start of CT infusion. Serum calcium decreased significantly 1 h after hormone administration and was accompanied by increased incidence of tetany. Serum phosphate levels were significantly lower than those of corresponding PTX controls 1 h post-CT, indicating that CT prevented the expected hyperphosphatemia. Higher CT dose (20 IU/h) in PTX birds substantially enhanced urine flow, CNa/CIn, CK/CIn, and occurrence of tetany.

Renal handling of phosphate, calcium, sodium, and potassium in intact and parathyroidectomized Rana pipiens.

Renal excretion of phosphate, calcium, sodium, and potassium in intact and parathyroidectomized male Rana pipiens was studied by renal clearance techniques using 14C-inulin. In intact frogs, 57% of filtered phosphate, 60% of filtered calcium, 97% of filtered sodium, and 89% of filtered potassium was reabsorbed by the renal tubules. Following parathyroidectomy, the rate of reabsorption of phosphate became significantly higher than that of the intact frog, and the relative phosphate clearance (fractional excretion) decreased. These changes corresponded with a gradual rise in serum phosphate values. There was no major effect on excretion patterns of calcium, sodium, or potassium after parathyroidectomy. These results suggest that in frogs the parathyroid glands strongly influence phosphate excretion patterns but have little effect on the excretion of calcium, sodium, or potassium.

Effects of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on embryonic chick bone in organ culture.

The ability of embryonic chick bone to respond to 25 hydroxyvitamin D3 and 1,25 dihydroxyvitamin D3 was assessed in organ culture. These metabolites were added to prelabeled chick embryo long-bone explants, and the amount of calcium-45 released into the medium after 24 or 48 h of hormone exposure was measured. For each time period a significant release of calcium-45 from hormone-treated bones was observed. The response to 25 hydroxyvitamin D3 and 1,25 dihydroxyvitamin D3 was always greater at 48 h and a clear dose-dependency was established at this time as well. 1,25 dihydroxyvitamin D3 was the more potent resorbing agent at all concentrations tested. The results from this study suggest that vitamin D metabolites may be involved in bone regulation in the chick embryo.

Renal excretion of phosphate and calcium in parathyroidectomized starlings.

Renal excretion patterns of calcium, phosphate, sodium, and potassium were studied in parathyroidectomized (PTX) and parathyroid extract (PTE)-injected PTX starlings. Sturnus vulgaris. Anesthetized birds (Equi-Thesin or Dial) were infused intravenously with 2.5% mannitol containing [14C]inulin. PTX caused significant hypocalcemia, hyperphosphatemia, increased relative calcium clearance (CCa/CIn), and decreased relative clearances of phosphate and potassium, but did not change the clearance of sodium. Glomerular filtration rate (GFR=CIn) and urine flow remained unchanged up to 2 h after PTX. PTE administration 3 h after PTX returned serum calcium and phosphate values to control levels and caused a transient (10-min) increase in GFR. Following PTE, the relative clearances of phosphate, sodium- and potassium increased, while that of calcium decreased significantly relative to the PTX levels. PTE caused net tubular secretion of phosphate, decreased tubular reabsorption of sodium and potassium (sometimes potassium secretion), and a return of excretion of calcium to control levels. These studies indicate that the parathyroid role in calcium and phosphate homeostasis in starlings is predominantly on the kidney.

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.

Parathyroid hormone effect on chicken renal brush-border membrane phosphate transport.

Renal clearance studies showed that parathyroidectomy (PTX) stimulated renal PO4 reabsorption within 90 min in 3-wk-old chicks. Subsequent parathyroid hormone (PTH) infusion caused net PO4 secretion. Kidneys were taken for brush-border membrane (BBM) preparations 3 h after PTX or sham operations and 1 h after PTH or saline injections, thus yielding four categories of PTH status: PTX/saline, sham/saline, PTX/PTH, and sham/PTH. Efficacy of PTX and PTH was confirmed by examination of serum Ca concentrations. A 100 mM NaSCN gradient, out greater than in, caused concentrative PO4 uptake by BBM from sham/saline animals. Concentrative uptake was not produced by 100 mM KSCN, out greater than in; pH 7.4in vs. pH 5.4out; or 100 mM NaC1, out = in. Removal of endogenous PTH (PTX/saline) significantly stimulated Na-dependent PO4 uptake compared with sham/saline. PTH infusion significantly depressed Na-dependent PO4 uptake in PTX/PTH and sham/PTH groups compared with sham/saline and PTX/saline. Na-dependent, concentrative glucose uptake was present and unchanged by PTH. Kinetic analysis, based on 5-s uptakes, showed that both apparent affinity and maximal velocity (Vmax) for Na-dependent PO4 transport were decreased by PTH treatment. The data suggest that PTH influences avian renal PO4 excretion at least in part through a Na-dependent PO4 transport system in proximal tubule BBM.