Relationship between thinning of eggshells and reduction in number of eggs produced after administration of some saluretic drugs in domestic fowls

1. The effect of some diuretic drugs (acetazolamide, hydrochlorothiazide, chlorthalidone, furosemide, ethacrynic acid and amiloride) on eggshell formation and egg production in domestic fowls were investigated. All of the tested compounds significantly inhibited eggshell formation, furosemide being the most potent drug.2. The number of laid eggs was also reduced by several of the diuretics. There was a correlation between reduction of shell thickness and number of eggs (r = 0.77; P < 0.01).3. If the tested diuretics were subdivided into two groups according to their different modes of action, where furosemide, ethacrynic acid and amiloride represent sodium transport inhibitors, and acetazolamide, hydrochlorothiazide and chlorthalidone (contributory action of the two latter) are inhibitors of carbanhydrase, the highest correlation coefficient (r = 0.96) between reduction of shell thickness and egg production was found for sodium transport inhibitors. The corresponding correlation coefficient for carbanhydrase inhibitors (r = 0.44) was not significant.4. The probable mechanisms of action of the diuretics on eggshell formation and ovulation are discussed.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

The effect of diuretics and vasopressin on the osmotic permeability of the frog urinary bladder.

1. Large concentrations (in mM) of ethacrynic acid (0.1), furosemide (1.0), theophylline (5.0) and osmotic diuretics (100.0) sharply increased the flux of water along an osmotic gradient through the frog urinary bladder wall. Spironolactone (0.1), and hydrochlorothiazide (5.0) showed only a weak action on osmotic permeability. MercusalR, clopamide and triamterene did not affect water transport. 2. The presence of 0.2--1.0 mU/ml vasopressin (ADH) after pretreatment with a diuretic did not result in summation of the effects of both drugs used. 0.01--0.1 mM ethacrynic acid and 0.01 mM MercusalR significantly decreased the reaction to ADH. 1.0 mM furosemide, 0.1 mM spironolactone, 0.01 mM clopamide and 0.8 mM acetazolamide did not change the reaction to ADH. A reduction in the cellular response to ADH and a decrease in the osmotic permeability of the tubular wall may be responsible in part for the diuretic action of ethacrynic acid and MercusalR.     

Interaction of amiloride and hydrochlorothiazide with atrial natriuretic factor in the medullary collecting duct

Medullary collecting duct function was studied using the in vivo microcatheterization technique in three groups of rats receiving amiloride, hydrochlorothiazide, or both diuretics. In each group of animals, atrial natriuretic factor (ANF99-126) was given in the second phase of the experiment. The combination of amiloride and hydrochlorothiazide resulted in a more marked natriuresis than either diuretic given as a single agent. Sodium reabsorption in the medullary collecting duct, as a fraction of the delivered load, was reduced from 64% (amiloride) and 69% (hydrochlorothiazide) to 29% (amiloride and hydrochlorothiazide). Atrial natriuretic factor reduced collecting duct sodium reabsorption when added to amiloride or hydrochlorothiazide to 23% and to 41%, respectively, but had no additional effect when given with amiloride and hydrochlorothiazide. Potassium excretion with amiloride and hydrochlorothiazide was intermediate between amiloride or hydrochlorothiazide given as single agents. With the diuretic combination, potassium transport showed no significant reabsorption or secretion along the medullary collecting duct, amiloride was associated with potassium reabsorption, and hydrochlorothiazide was associated with potassium secretion in the duct. The results confirm the importance of the medullary collecting duct as a site of diuretic action. The known additive effects of amiloride and hydrochlorothiazide on sodium excretion and the opposing effects of these agents on potassium excretion occur, to a major degree, in the medullary collecting duct. Furthermore, the additive effects of amiloride and ANF indicate that blocking of amiloride-sensitive sodium channels is not the only mechanism of action of ANF on duct salt transport in vivo.     

Reduction of the effects of diuretics or sodium chloride loading in the conscious rat by inhibitors of prostaglandin synthesis

In conscious rats pretreatment with indomethacin or flurbiprofen, two chemically unrelated inhibitors of prostaglandin synthesis, reduced urine volume and sodium excretion induced by four diuretics, acetazolamide, amiloride, bendrofluazide and frusemide, or oral sodium chloride loads. The maximum reduction in sodium excretion was limited to approximately 2 mmol/kg Na⁺ even when sodium excretion was greatly increased. In contrast these inhibitors did not appreciably affect potassium excretion. These results indicate that part of the natriuretic response in the rat to highly and moderately efficacious diuretics and to sodium chloride loading is modified by prostaglandins. We suggest that the lack of effects on potassium excretion indicate that the collecting tubule is the probable site of action.     

Carbonic anhydrase inhibitors. Comparison of chlorthalidone,indapamide, trichloromethiazide,and furosemide X-ray crystal structures in adducts with isozyme II,when several water molecules make the difference

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K_Is of 65–138 nM), whereas indapamide is a much weaker one (K_I of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2–3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.     

Uterine and eggshell structure and histochemistry in a lizard with prolonged uterine egg retention (Lacertilia,Scincidae, Saiphos)

The eggshell of lizards is a complex structure composed of organic and inorganic molecules secreted by the oviduct, which protects the embryo by providing a barrier to the external environment and also allows the exchange of respiratory gases and water for life support. Calcium deposited on the surface of the eggshell provides an important nutrient source for the embryo. Variation in physical conditions encountered by eggs results in a tradeoff among these functions and influences eggshell structure. Evolution of prolonged uterine egg retention results in a significant change in the incubation environment, notably reduction in efficiency of gas exchange, and selection should favor a concomitant reduction in eggshell thickness. This model is supported by studies that demonstrate an inverse correlation between eggshell thickness and length of uterine egg retention. One mechanism leading to thinning of the eggshell is reduction in size of uterine shell glands. Saiphos equalis is an Australian scincid lizard with an unusual pattern of geographic variation in reproductive mode. All populations retain eggs in the uterus beyond the embryonic stage at oviposition typical for lizards, and some are viviparous. We compared structure and histochemistry of the uterus and eggshell of two populations of S. equalis, prolonged egg retention, and viviparous to test the hypotheses: 1) eggshell thickness is inversely correlated with length of egg retention and 2) eggshell thickness is positively correlated with size of shell glands. We found support for the first hypothesis but also found that eggshells of both populations are surprisingly thick compared with other lizards. Our histochemical data support prior conclusions that uterine shell glands are the source of protein fiber matrix of the eggshell, but we did not find a correlation between size of shell glands and eggshell thickness. Eggshell thickness is likely determined by density of uterine shell glands in this species. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Effects of experimental calcium availability,egg parameters and laying order on Great Tit Parus major eggshell pigmentation patterns

Many bird species lay eggs speckled with protoporphyrin‐based spots, however, for most of them the function of eggshell spotting is unknown. A plausible hypothesis is that protoporphyrin might have a structural function in strengthening the eggshell and is therefore deposited when calcium is scarce. In this study, we experimentally provided Great Tit Parus major females with supplemental calcium to examine its effect on the protoporphyrin‐based maculation of their eggs. In addition, we studied variation in eggshell pigmentation patterns in relation to other egg parameters and laying order. Calcium‐supplemented females laid larger eggs but shell thickness was not significantly affected by the treatment. Calcium supplementation may reduce the time and energy females devote to searching for calcium‐rich material, so that they can collect more nutrients and so lay larger eggs. Furthermore, pigment darkness was associated with egg volume and shape, which suggests that female quality and environmental food availability may also influence the shell pigmentation pattern. Within clutches, later‐laid eggs had larger and darker spots that were distributed more unevenly on the shell surface. This within‐clutch pattern could be explained by the increase in egg volume and egg shape and a decline in shell thickness with egg‐laying order, which characteristics were all related to shell‐spotting pattern. Eggs with a coronal ring had thinner shells, but pigment intensity and spot size were not related to shell thickness. Thus, our results suggest that concentrated spotting distribution may have a mechanical function, supporting the structural‐function hypothesis.     

Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

The in vitro and in vivo regulation of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of [3H]Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approximately equal to 30 - 130 microM). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki = 100-1000 microM). Administration of furosemide to rats for five days resulted in a profound diuresis (approximately equal to 350% increase in urine volume) accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of [3H]Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors.     

Antihypertensive and bilateral renal responses to diuretics in 2-kidney, 1-clip Goldblatt hypertensive rats

Experiments were conducted to assess the effect of furosemide or amiloride alone and a combination of both agents on each kidney in anesthetized 2-kidney, 1 clip Goldblatt hypertensive rats (n = 25). Intravenous infusion of furosemide alone (1.02 mg/kg.hr) significantly reduced the blood pressure by 14 +/- 5 mmHg. There were 6- to 10-fold increases in water, absolute sodium and fractional sodium excretions and a 2-fold increase in potassium excretion in the nonclipped kidney. A smaller but significant increase in the excretory function was also observed in the clipped kidney. There was no significant change in GFR of both kidneys. Indomethacin pretreatment (2 mg/kg) failed to significantly alter the vasodepressor and renal responses to furosemide in both hypertensive and normal rats. Removal of the renal artery clip from the hypertensive rats reduced the blood pressure by 12 +/- 3 mmHg and enhanced the function of the ipsilateral, unclipped kidney. Subsequent administration of furosemide further increased the excretory response. Administration of amiloride alone (2.4 mg/kg.hr) or with furosemide into hypertensive rats reduced the arterial pressure and increased excretion rates of urine flow and urinary sodium. Potassium excretion rate decreased bilaterally in amiloride treated rats but did not alter significantly in rats which received a combination of amiloride and furosemide. These results indicate that diuretics ameliorate the excretory function of both the stenotic kidney and the nonstenotic kidney and that the improvement of the kidney function is independent of prostaglandin. Furthermore, removal of the stenosis accentuates the beneficial effect of diuretics on the kidney.     

Effects of anion and cation inhibitors and carbonic anhydrase inhibitors upon the activity of the gypsy moth (Lepidoptera: Lymantriidae) nucleo-polyhedrovirus

Twenty chemicals that were reported to act as anion transport inhibitors, cation transport inhibitors, and inhibitors of carbonic anhydrase activity were tested at a 1% concentration (wt:wt) for their effects upon the biological activity of the gypsy moth, Lymantria dispar (L.), nucleopolyhedrovirus (LdMNPV). Among the five anion transport inhibitors tested, flufenamic acid acted as a viral enhancer. None of the seven inhibitors of K+ enhanced viral activity and three (4-aminopyridine, diacetyl, and procaine) significantly reduced the activity of LdMNPV. All four Na+ transport inhibitors (abietic acid, amiloride, dicyclohexylcarbodiimide, triamterene) acted as viral enhancers. Triamterene was the most active enhancer, as the LC50 was reduced by approximately 1,750-fold. Five carbonic anhydrase inhibitors were tested and four (acetazolamide, hydrochlorothiazide, methazolamide, sulfanilamide) enhanced the activity of LdMNPV. Acetazolamide (a carbonic anhydrase inhibitor), amiloride (a Na+ transport inhibitor), and flufenamic acid (an anion transport inhibitor) were tested singly and in different combinations. Every combination tested (acetazolamide/amiloride, acetazolamide/flufenamic acid, amiloride/flufenamic acid, acetazolamide/amiloride/flufenamic acid) significantly decreased the LC50 from 7.79 PIB/mm2 to a value as low as 0.008 PIB/mm2 (amiloride/flufenamic acid).     

Effect of single dose of diuretics on renal magnesium excretion in man, with special reference to their site of action.

The administration of a single dose of furosemide, ethacrynic acid and polythiazide to healthy individuals under conditions of maximum water diuresis produces a significant increase in renal magnesium excretion. Elevated Mg excretion displayed a direct correlation to renal sodium excretion after furosemide (r=0.689, p less than 0.001), ethacrynic acid (r=0.869, p less than 0.001) and polythiazide (r=0.586, p less than 0.01). The slopes of the various regression lines did not differe significantly from each other or from the slope of the regression line characterizing this correlation for mannitol (r= 0.603, p less than 0.01). A significant linear correlation was likewise found between the excretion of Mg and total osmotically active substances after furosemide (r=0.783, p less than 0.001), ethacrynic acid (r=0.88, p less than 0.001) and polythiazide (r=0.646, p less than 0.01). The regression lines of the given correlations did not differ significantlyfrom each other, but their slopes were significantly higher than that of the regression line for the correlation after mannitol (r=0.454, p less than 0.01). The findings indicate that tubular Mg transport is influenced both by a decrease in tubular Na resorption in the diluting segment (polythiazide) and by an effect on Na resorption in the parts of the nephron proximal to the diluting segment of the nephron (furosemide, ethacrynic acid).     

Magellanic Penguin eggshell pores: does number matter?

Most penguin species lay two eggs 3–4 days apart that hatch 1–2 days apart. Hypotheses to explain the shorter incubation period for the second egg include differences in porosity of the eggshells. Eggs with more or larger pores and/or thinner shells have higher gas exchange rates and faster embryonic development. We used eggshells from hatchling Magellanic Penguins Spheniscus magellanicus to test whether eggshell thickness and pore density affect incubation period. We expected: (i) second eggs to have thinner shells and/or more pores than first eggs; (ii) eggs to have thinner shells and/or more pores if they were laid by older females, later in the season, or in burrow nests (more humid than open nests under bushes). We found no support for these hypotheses. Egg pores and shell thickness were not related to incubation period. Eggs from the same clutch were similar in eggshell thickness and pore density. Female age, laying date and nest type were poor predictors of eggshell thickness and pore density. Egg pores, we conclude, have little explanatory power for differences in incubation time of eggs in Magellanic Penguins, and suggest that synchronous onset of incubation matters.     

产卵次序对山麻雀卵色、卵大小和重量的影响

产卵次序可对鸟类卵黄中的营养成分、卵壳色素以及卵壳厚度等产生影响。一些鸟类可通过产卵次序对不同卵的资源进行分配,从而实现繁殖成效的最大化。本研究对不同产卵次序下山麻雀(Passer cinnamomeus)的卵色、卵大小和卵重量进行分析,结果表明,山麻雀的卵重和卵大小随着产卵次序并无显著变化,卵色与卵的大小和重量均无显著相关性,但卵背景的色度随产卵次序有明显下降趋势,而卵斑点密度则呈相反的变化。卵背景的色度随产卵次序下降可能是山麻雀有限的色素在卵中不均匀分配的结果,而卵斑点密度的增加则可能是对后期卵壳厚度变薄的一种补偿。     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Effects of ethacrynic acid and furosemide on phosphorylation reactions of kidney mitochondria. Inhibition of the adenine nucleotide translocase.

Previous reports that ethacrynic acid and furosemide diminish mitochondrial P : O ratios and reduce (Na+ + K+)-ATPase activity suggested that these diuretics may inhibit mitochondrial phosphorylation reactions. This possibility was initially studied by determining the effects of ethacrynic acid and furosemide on [32P]ATP exchange activity of rat kidney mitochondria. Concentrations of both drugs at 10(-4) M or greater, significantly inhibited [32P]ATP exchange. To investigate the mechanism of this inhibition, the effects of ethacrynic acid and furosemide on the ATPase activity of intract mitochondria and sonicated submitochondrial particles were determined. Both diuretics inhibited ATPase activity of intact mitochondria at 10(-4) M. In contrast, ATPase of submitochondrial particles was significantly less susceptible to inhibition by the diuretics. These results suggested that ethacrynic acid anf furosemide inhibit adenine nucleotide transport across the mitochondrial membrane. This was directly tested by determining the effects of the diretics on the mitochondrial adenine nucleotide translocase. At 5-10(-4) M, both ethacrynic acid and furosemide significantly inhibited adenine nucleotide transport. These findings suggest that ethacrynic acid and furosemide may diminish renal tubular solute reabsorption by direct inhibition of adenine nucleotide transport across the mitochondrial inner membrane.     

Influence of the evolution of viviparity on eggshell morphology in the lizard Lerista bougainvillii

In reptiles, the evolutionary transition from egg-laying to live-bearing is thought to involve a gradual increase in the duration of egg retention, with progressively more development occurring prior to oviposition, and culminating in the birth of fully developed offspring. However, prolonging the retention of fully-shelled eggs within the oviducts may pose serious gas-exchange problems for the embryos. Thus, evolutionary increases in the period of intrauterine retention may require correlated decreases in the thickness of eggshells and/or their degree of calcification to allow for adequate embryonic gas exchange. To test this evolutionary model, eggs of three distinct reproductive forms of the scincid lizard Lerista bougainvillii were examined to determine the evolutionary relationships between the thickness of the shell membrane, degree of eggshell calcification, and the duration of uterine egg retention. These comparisons revealed the predicted pattern of correlated shifts in eggshell morphology and embryonic stage at oviposition. Evolutionary increases in the duration of egg retention were accompanied by decreases in the thickness of the eggshell membrane and degree of eggshell calcification. This evolutionary model suggests that there may be a tradeoff between the advantages of extended egg retention and the disadvantages of a thinner eggshell. On the basis of this tradeoff, I propose that oviparous taxa with relatively thin eggshells may be preadapted to evolve viviparity. Comparative examination of the limited data available on eggshell thickness in lizards supports this possibility. © 1996 Wiley-Liss, Inc.     

Inhibition of renal PGE2-9-ketoreductase by diuretics

The metabolism of PGE₂ by extracts of renal cortex is species dependent. In the rat PGE₂-15-hydroxydehydrogenase initiates metabolism whereas in the rabbit PGE₂-9-ketoreductase predominates. In man both mechanisms may operate. Each of the metabolic enzymes, which limits the vasodilator-diuretic actions of PGE₂, was inhibited by ethacrynic acid, furosemide and indomethacin. Some inhibition of PGE₂-9-ketoreductase was also observed with chlorthalidone, hydralazine and phentolamine but the thiazide diuretics and a number of other cardiovascular-active agents were without significant effect. We conclude that the inhibition of PGE₂-9-ketoreductase and PGE₂-15-hydroxydehydrogenase could contribute to the mechanism of action of the non-thiazide diuretics in man.     

Selenium distribution in eggs of avian species

We studied the effect of egg mass of eight different avian species on Se distribution between egg components and the effect of incubation on Se accumulation by chicken eggshell and shell membrane. Eight groups of birds received a diet without Se supplementation. Unfertile eggs were collected after 35 days of feeding; yolk, albumen, shell and shell membrane were assayed separately for Se. All avian species studied showed identical Se concentration in yolk–albumen complex equal to 38.7 μg Se/100 g, reflecting a linear correlation between yolk–albumen mass and Se content. Shells and shell membrane Se accumulation showed quadratic correlation with the appropriate mass thus explaining unusually high Se concentration in ostrich shell and shell membrane, that reached values 1785 and 1904 μg Se/kg respectively. Incubation of fertile eggs decreased eggshell Se content, the effect being more expressed in eggs from hens fed sodium selenite compared to organic Se utilization (Sel-Plex). It was concluded that shell might be an additional Se source for an embryo.     

Evaluation of a protocol for studying the chick chorioallantoic membrane in vitro

Explants of eggshell with and without the chorioallantoic membrane were taken from fertile chicken eggs on day 16 of incubation and exposed in vitro to inhibitors (acetazolamide and benzolamide) of carbonic anhydrase to determine if enzyme inhibition affected release of calcium from the shell. A separate experiment examined the effect of the metabolic poison dinitrophenol (DNP) on release of calcium from explants. Explants with the chorioallantois in situ released more calcium than those lacking the epithelium, but neither the enzyme inhibitors nor DNP affected release of calcium. The lack of effect of the enzyme inhibitors could indicate that activity of carbonic anhydrase is not as important to the release of calcium from the eggshell as has been assumed. However, the absence of an effect of DNP instead indicates that release of calcium mediated by the chorioallantois in vitro simply lacks physiological relevance. Thus, results of this investigation raise doubts that the mechanism underlying release of calcium from the eggshell can be assessed in vitro.