Mitochondrial Membrane Surface Area Research Articles

Protonic Bioenergetics in Mitochondria and Neurons

For decades, it was not entirely clear why mitochondria develop cristae? The work employing the transmembrane-electrostatic proton localization theory reported here has now provided a clear answer to this fundamental question. Surprisingly, the transmembrane-electrostatically localized proton concentration at a curved mitochondrial crista tip can be significantly higher than that at the relatively flat membrane plane regions where the proton-pumping respiratory supercomplexes are situated. The biological significance for mitochondrial cristae has now, for the first time, been elucidated at a protonic bioenergetics level: 1) The formation of cristae creates more mitochondrial inner membrane surface area and thus more protonic capacitance for transmembrane-electrostatically localized proton energy storage; and 2) The geometric effect of a mitochondrial crista enhances the transmembrane-electrostatically localized proton density to the crista tip where the ATP synthase can readily utilize the localized proton density to drive ATP synthesis. Accordingly, the neural resting/action potential is essentially a protonic/cationic membrane capacitor behavior. Neural resting and action potential is now much better understood as the voltage contributed by the localized protons/cations at a neural liquid- membrane interface. The newly formulated action potential equation provides biophysical insights for neuron electrophysiology, which may represent a complementary development to the classic Goldman-Hodgkin-Katz equation.

Morphometric Analysis of the Internal Ultrastructure of Mitochondria of Muscle Tissue in Horsehair Worm Gordionus alpestris (Nematomorpha)

Internal ultrastructure of the muscle tissue mitochondria of horsehair worm Gordionus alpestris (Nematomorpha) was studied using morphometry. Surface area of the inner mitochondrial membrane per unit of the mitochondrial volume, or surface density of the inner mitochondrial membrane, was measured as a main morphometric parameter. The surface density of the inner mitochondrial membrane of the G. alpestris muscle tissue was compared to the respective parameter of the skeletal and cardiac muscle mitochondria. The surface density of the inner mitochondrial membrane of the worm was close to the surface density values of the cardimyocytes of 3-month-old mice and Wistar rats and was slightly higher than the surface density of mitochondria from the skeletal muscle of 3-month-old mice. The functional significance of the well-developed system of mitochondrial membranes of extended mitochondria of the horsehair worm is discussed as a structure necessary to ensure effective functioning of the circomyarian conntractile apparatus in the muscle tissue of the horsehair worm.

Protonic Capacitor: Elucidating the biological significance of mitochondrial cristae formation

For decades, it was not entirely clear why mitochondria develop cristae? The work employing the transmembrane-electrostatic proton localization theory reported here has now provided a clear answer to this fundamental question. Surprisingly, the transmembrane-electrostatically localized proton concentration at a curved mitochondrial crista tip can be significantly higher than that at the relatively flat membrane plane regions where the proton-pumping respiratory supercomplexes are situated. The biological significance for mitochondrial cristae has now, for the first time, been elucidated at a protonic bioenergetics level: 1) The formation of cristae creates more mitochondrial inner membrane surface area and thus more protonic capacitance for transmembrane-electrostatically localized proton energy storage; and 2) The geometric effect of a mitochondrial crista enhances the transmembrane-electrostatically localized proton density to the crista tip where the ATP synthase can readily utilize the localized proton density to drive ATP synthesis.

Scaling of morphology and ultrastructure of hearts among wild African antelope.

The hearts of smaller mammals tend to operate at higher mass-specific mechanical work rates than those of larger mammals. The ultrastructural characteristics of the heart that allow for such variation in work rate are still largely unknown. We have used perfusion-fixation, transmission electron microscopy and stereology to assess the morphology and anatomical aerobic power density of the heart as a function of body mass across six species of wild African antelope differing by approximately 20-fold in body mass. The survival of wild antelope, as prey animals, depends on competent cardiovascular performance. We found that relative heart mass (gkg-1 body mass) decreases with body mass according to a power equation with an exponent of -0.12±0.07 (±95% confidence interval). Likewise, capillary length density (kmcm-3 of cardiomyocyte), mitochondrial volume density (fraction of cardiomyocyte) and mitochondrial inner membrane surface density (m2cm-3 of mitochondria) also decrease with body mass with exponents of -0.17±0.16, -0.06±0.05 and -0.07±0.05, respectively, trends likely to be associated with the greater mass-specific mechanical work rate of the heart in smaller antelope. Finally, we found proportionality between quantitative characteristics of a structure responsible for the delivery of oxygen (total capillary length) and those of a structure that ultimately uses that oxygen (total mitochondrial inner membrane surface area), which provides support for the economic principle of symmorphosis at the cellular level of the oxygen cascade in an aerobic organ.

Symmorphosis and the insect respiratory system: allometric variation

Taylor and Weibel's theory of symmorphosis predicts that structures of the respiratory system are matched to maximum functional requirements with minimal excess capacity. We tested this hypothesis in the respiratory system of the migratory locust, Locusta migratoria, by comparing the aerobic capacity of the jumping muscles with the morphology of the oxygen cascade in the hopping legs using an intraspecific allometric analysis of different body mass (M(b)) at selected juvenile life stages. The maximum oxygen consumption rate of the hopping muscle during jumping exercise scales as M(b)(1.02±0.02), which parallels the scaling of mitochondrial volume in the hopping muscle, M(b)(1.02±0.08), and the total surface area of inner mitochondrial membrane, M(b)(0.99±0.10). Likewise, at the oxygen supply end of the insect respiratory system, there is congruence between the aerobic capacity of the hopping muscle and the total volume of tracheoles in the hopping muscle, M(b)(0.99±0.16), the total inner surface area of the tracheoles, M(b)(0.99±0.16), and the anatomical radial diffusing capacity of the tracheoles, M(b)(0.99±0.18). Therefore, the principles of symmorphosis are upheld at each step of the oxygen cascade in the respiratory system of the migratory locust.

Mammalian metabolism: insights from arid zone reptiles.

Mammals, being endotherms have very high metabolic rates compared to ectothermic reptiles. Similarly, small mammals have high rates of mass-specific metabolism compared to larger mammals. This review examines the mechanistic basis of why particular mammal species have a specific metabolic rate. Initial studies compared mammals with arid zone reptile species of the same size and Tb. Mammals have larger internal organs, with more mitochondrial membrane surface area than the reptiles. The cells of mammals are leakier to Na+ ions and their mitochondrial membranes are leakier to H+ ions than in reptile cells. These leakier membranes have membrane lipids that are polyunsaturated and less monounsaturated than their less leaky counterparts. Examination of the cellular basis of allometric variation in metabolism in mammals reveals very similar findings with polyunsaturated membranes associated with the high mass-specific metabolic rates of small mammal species and monounsaturated membranes with low rates of metabolism of large mammals. These findings have resulted in the development of the ?membrane pacemaker? theory of metabolism, which proposes that membrane bilayer composition is regulated in animals and that highly polyunsaturated membranes result in enhanced molecular activity of membrane proteins and in turn this results in an elevated metabolic rate of cells, tissues and consequently whole animals. This theory is also supported by the recent examination of the basis of body-size variation in the metabolic rates of birds. The ?membrane pacemaker? theory of metabolism is currently the only explanation of the mechanisms determining the metabolic rate and thus the cost of living of animals. It has implications for the effect of food habits on metabolism and the relationship between metabolism and lifespan.

Allometry of mammalian cellular oxygen consumption.

In the 1930s, Max Kleiber and Samuel Brody established that the interspecies correlation between mammalian body mass and metabolic rate (alphaM(0.75)) cannot be explained (solely) by whole body surface area (alphaM(0.66)) to volume ratios. Metabolic considerations must also be taken into account. Decreases in the proportion of visceral organ mass to whole body mass can account for some of the whole body metabolic differences. However, superimposed upon these anatomical differences, the metabolism of tissues and cells has been demonstrated to decrease with increasing body mass. These decreases in oxygen consumption rates (with increasing body mass) in cells and tissues can be explained by a decrease in ATP turnover and mitochondrial density and an increase in mitochondrial functional efficiency (decrease in proton leak). The majority of the proton leak differences reflect differences in mitochondrial inner membrane surface area. Indeed, liver metabolism correlates directly with liver mitochondrial inner membrane surface area. Apart from being a significant contributor (approximately 25%) to basal metabolism, mitochondrial proton leak is a major factor determining the differences in basal metabolism between mammals of different body mass.

The interplay among cardiac ultrastructure, metabolism and the expression of oxygen-binding proteins in Antarctic fishes.

We examined heart ventricle from three species of Antarctic fishes that vary in their expression of oxygen-binding proteins to investigate how some of these fishes maintain cardiac function despite the loss of hemoglobin (Hb) and/or myoglobin (Mb). We quantified ultrastructural features and enzymatic indices of metabolic capacity in cardiac muscle from Gobionotothen gibberifrons, which expresses both Hb and Mb, Chionodraco rastrospinosus, which lacks Hb but expresses Mb, and Chaenocephalus aceratus, which lacks both Hb and Mb. The most striking difference in cellular architecture of the heart among these species is the percentage of cell volume occupied by mitochondria, V(v)(mit,f), which is greatest in Chaenocephalus aceratus (36.53+/-2.07), intermediate in Chionodraco rastrospinosus (20.10+/-0.74) and lowest in G. gibberifrons (15.87+/-0.74). There are also differences in mitochondrial morphologies among the three species. The surface area of inner mitochondrial membrane per volume of mitochondria, S(v)(imm, mit), varies inversely with mitochondrial volume density so that S(v)(imm,mit) is greatest in G. gibberifrons (29.63+/-1.62 microm(-)(1)), lower in Chionodraco rastrospinosus (21.52+/-0.69 microm(-)(1)) and smallest in Chaenocephalus aceratus (20.04+/-0.79 microm(-)(1)). The surface area of mitochondrial cristae per gram of tissue, however, is greater in Chaenocephalus aceratus than in G. gibberifrons and Chionodraco rastrospinosus, whose surface areas are similar. Despite significant ultrastructural differences, oxidative capacities, estimated from measurements of maximal activities per gram of tissue of enzymes from aerobic metabolic pathways, are similar among the three species. The combination of ultrastructural and enzymatic data indicates that there are differences in the density of electron transport chain proteins within the inner mitochondrial membrane; proteins are less densely packed within the cristae of hearts from Chaenocephalus aceratus than in the other two species. High mitochondrial densities within hearts from species that lack oxygen-binding proteins may help maintain oxygen flux by decreasing the diffusion distance between the ventricular lumen and mitochondrial membrane. Also, high mitochondrial densities result in a high intracellular lipid content, which may enhance oxygen diffusion because of the higher solubility of oxygen in lipid compared with cytoplasm. These results indicate that features of cardiac myocyte architecture in species lacking oxygen-binding proteins may maintain oxygen flux, ensuring that aerobic metabolic capacity is not diminished and that cardiac function is maintained.

Potential definition of the time of death from autolytic myocardial cells:: a morphometric study

Morphometric methods and transmission electron microscopy were used to quantify modifications occurring in the mitochondria of dog myocardium during the first four hours of autolysis. Myocardial fragments were obtained from the outer free wall of the left ventricle, during anesthesia (control-zero) and at 15, 45, 120, and 240 min after cardiac arrest, maintaining the heart “in situ” at 22°C. During the 240 min of autolysis, the main parameters evaluated showed: (a) a decrease in the number of mitochondria from 0.31 to 0.12 per μm 3 of cytoplasm. The decrease over the first 45 min reached 50% of the initial value; (b) an increase in mitochondrial volume, three times greater after the first 45 min (from 0.92 to 2.68 μm 3) and four times greater after 240 min (from 0.92 to 3.79 μm 3); (c) an increase in mitochondrial outer membrane surface area from 5.51 to 12.54 μm 2; (d) an increase in the surface area of individual mitochondria inner membrane and cristae from 27.60 to 56.96 μm 2. The progressive nature of the alterations and the difference in the numerically expressed values allow correlation with the time of somatic death. The authors emphasize the need for further studies in order to complement the present study.

Energetics of metamorphosis in two holometabolous insect species:Manduca sexta (Lepidoptera: Sphingidae) andTenebrio molitor (Coleoptera: Tenebrionidae)

Gas-exchange rates (VO2 and VCO2) measured from intact Manduca sexta and Tenebrio molitor pupae throughout metamorphosis decrease immediately following pupation, then increase during the last 60% of pupal development. Metabolic rates and rate of development increased with temperature between 22°C and 32°C in both species. There was, however, no significant effect of temperature on cumulative oxygen consumption over the duration of metamorphosis for either species. Although M. sexta pupae are 10 times as large as T. molitor pupae, their mass-specific cost of metamorphosis (per gram of pharate adult) was similar to that of T. molitor. The large-scale histolysis and subsequent remodeling of tissues during metamorphosis makes it difficult to quantify the mass of metabolically active tissue in pupae. A mitochondrial index (MI) related to mitochondrial membrane surface area was calculated using rhodamine-123-labeled mitochondria from M. sexta pupae, yielding a measure of pupa oxidative capacity and, indirectly, the amount of active tissue in the pupa. Across development, MI fluctuated in a manner resembling VO2 curves from intact M. sexta. Although both MI and VO2 increased during the latter half of development, the increase in MI began earlier and was proportionally larger than the observed increase in VO2. This suggests that, through most of metamorphosis, M. sexta pupae probably have a substantial aerobic potential that remains unutilized until emergence of the adult moth. J. Exp. Zool. 280:344–353, 1998. © 1998 Wiley-Liss, Inc.

Sentinels of Leydig cell structural and functional changes in golden hamsters in early testicular regression and recrudescence.

The seasonally breeding hamster model was used to assess changes associated with Leydig cell activity and inactivity. Specifically, parameters of Leydig cell structure and function were studied to determine the early changes seen in seasonally breeding golden hamsters in photoperiod-induced gonadal regression and photoperiod-induced gonadal recrudescence. Time intervals used to characterize early regression and recrudescence were selected with the objective of determining the most sensitive parameters characterizing functional transitional states. In early regression, plasma levels of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH) or testosterone were reduced significantly. Regressive structural changes included decreases in volume of the interstitium, total number of Leydig cells, blood vessels, and the seminiferous tubules and tubular lumen. A decrease in volume, but not numbers of Leydig cells, was accompanied by decreases in volume of Leydig cell tubular smooth endoplasmic reticulum (t-ER), Golgi complex, and the peroxisomes and decreases in surface area of the inner mitochondrial membrane and t-ER, suggesting that early Leydig cell changes are restricted primarily to organelles associated with steroid biosynthesis. In early recrudescence, at a time when there was an increase in the number of germ cells in the basal compartment, blood levels of LH, FSH and testosterone were all increased. There were increases in testicular weight, volume, seminiferous tubular lumen, blood vessel and interstitial volumes. Leydig cells increased in size as a result of increases in nuclear, nucleolar and cytoplasmic volumes, while Leydig cell numbers did not increase. At the subcellular level there were increases in the surface areas of the cell, mitochondrial membranes and cisternal endoplasmic reticulum (sparsely populated with ribosomes). Unlike the changes seen in early degeneration when steroid synthetic organelles were initially affected, the changes in early recrudescence indicate that Leydig cells must first rebuild their synthetic machinery (nucleus, nucleolus and rough endoplasmic reticulum) that, at a later time, will reconstitute the cell's steroidogenic machinery. Correlation of hormonal parameters with structural parameters did indicate a relationship between hormonal parameters and steroid secreting organelles. Correlations were strongest with testosterone but, surprisingly, plasma FSH levels correlated more strongly with many structural parameters of the Leydig cell than did the levels of LH. Since FSH receptors are not present on Leydig cells, these data add to the growing data suggesting a role for factors originating from the seminiferous tubule in modulation of Leydig cell function.

Estimation of the oxygen gradient across phospholipid bilayers of mitochondria from reperfused rabbit hearts after ischemia.

Mitochondria isolated from myocardium exposed to 30 minute ischemia followed by 30 minute reperfusion showed an increase in membrane viscosity and a decrease in wobbling angle of phospholipids, compared with those from the normally perfused myocardium in anesthetized open-chest rabbits. The values for the membrane viscosity were used to estimate the oxygen gradient across the lipid bilayers of mitochondrial membranes with a model of cylindrical diffusion. The effective diffusion coefficient for oxygen, DO2, was approximated to be 6.5 and 6.3 x 10(-5) cm2/sec in the control and ischemic-reperfused area, respectively, by comparing reported DO2 values with values for membrane viscosity. For the surface area of the inner mitochondrial membrane including cristae and for the oxygen consumption rate of the myocardium, reported values for rats and cats, respectively, were employed. Using these values, oxygen gradients across the lipid bilayers of mitochondrial lipid membranes were estimated to be only 0.055 and 0.057 nM in the control and 30 minute ischemic-reperfused myocardium, respectively. If the mitochondrial membranes are hydrated because of the ischemia-reperfusion, the absorption coefficient of the membrane to oxygen will decrease and the oxygen gradient will be increased. In the present study, however, the fluorescence life time of DPH, the hydrophobic fluorophore, showed no shortening despite the ischemia-reperfusion. Hence, no indication of membrane hydration was obtained.

Development of mammalian endothermic metabolism: quantitative changes in tissue mitochondria

The development of energy metabolism of mammalian tissues was assessed in the tammar wallaby Macropus eugenii by the measurement of mitochondrial parameters in the liver, heart, kidney, and brain. Tissues taken from wallabies (n = 27) ranging from 10-day-old pouch young (weighing approximately 4 g) to adults (averaging 6.2 kg) were weighed and fixed, and mitochondrial volume and mitochondrial membrane surface area (MMSA) were determined by quantitative electron microscopy techniques. Developmental changes in these parameters were analyzed chronologically and allometrically. Relative growth rates of all four tissues decreased during development. Liver and heart showed constant allometric growth throughout development, whereas kidney and brain showed biphasic allometric growth. Tissue metabolic intensity assessed by MMSA (m2/cm3 tissue) was constant in liver, showed a threefold increase in brain during pouch life, showed a fourfold increase in the heart between 100 and 200 days of age, and showed a twofold increase in the kidney at the end of pouch life. In all tissues, adult levels of tissue metabolic capacity were present at pouch exit. In all four tissues, total MMSAs were at "reptilian" levels at birth and gradually increased to "mammalian" levels. Each tissue exhibited a different developmental timetable. When the total MMSAs for all four tissues were summed there was a similar pattern of allometric development between summed MMSA and whole animal metabolic rate.

Mitochondrial structure and function in CCl4-induced cirrhosis in the rat

To investigate whether the impairment of mitochondrial function in cirrhosis is due to a reduction in liver cell mass or whether mitochondrial function is altered specifically, we analyzed mitochondrial volume and surface density of mitochondrial membranes in control and cirrhotic rats by stereological means. Cirrhosis was induced by long-term exposure to phenobarbital and CCl4. Hepatocellular and mitochondrial volumes were reduced to a similar extent, by 39% and 40%, respectively, in cirrhotic animals (p less than 0.01). Thus the fraction of hepatocytes occupied by mitochondria did not differ between the two groups. Both total outer (31 +/- 3 vs. 19 +/- 6 m2; p less than 0.01) and inner (87 +/- 24 vs. 45 +/- 12 m2; p less than 0.01) mitochondrial membranes were significantly reduced. Membrane surface was normal per unit of mitochondrial volume, however, suggesting intact mitochondrial structure. Matrix and outer membrane enzyme activities expressed per compartment did not differ between control and cirrhotic animals. Inner membrane, in contrast, had an increased enzyme content per unit area both for cytochrome oxidase (10.3 +/- 2.9 vs. 13.0 +/- 1.6; p less than 0.05) and ATPase (13.7 +/- 1.4 vs. 21.2 +/- 2.9; p less than 0.01). Basal oxygen consumption measured in the perfused liver in situ was significantly reduced in cirrhotic livers (1.6 +/- 0.1 vs. 1.1 +/- 0.4 mumol/min-1/gm-1) but was unchanged when expressed per square meter of inner membrane. Our results demonstrate that impaired mitochondrial function is mainly due to loss of hepatocellular mass. Increased enzyme activity per unit surface area of inner mitochondrial membrane may be important to maintain mitochondrial function of the cirrhotic liver.

Cellular Responses of the Rat Adrenal Zona Fasciculata to Acute Acth Stimulation: A Morphometric Study

Long-term ACTH-stimulation of steroidogenesis in the rat adrenal cortex results in time-dependent increases in the surface area per cell of smooth endoplasmic reticulum and mitochondrial cristae. As the morphological responses to short-term ACTH stimulation have not been described, we undertook morphometric analyses of the effects of acute (10 min) ACTH stimulation of rat adrenocortical cells in vivo as they may be expressed in the mitochondria and the smooth endoplasmic reticulum. Six young male Wistar rats were allocated to each of four groups: 1. normal controls; 2. ACTH-treated normal rats; 3. Dexamethasone-inhibited; 4. ACTH-treated Dexamethasone-inhibited. As judged by the radio-immunoassay of trunk blood, levels of ACTH, 11-deoxycorticosterone and corticosterone were appropriate to the treatment state. ACTH activation resulted in no changes in the smooth endoplasmic reticulum; but the mitochondrial inter-membrane space was significantly increased over that of the contrasted pair. The inter-membrane space in the dexamethasone-inhibited rats was significantly less than that of all other groups. No responses to ACTH-activation were shown by the intra-cristal or matrix volumes of the mitochondria. The increased inter-membrane space appears to be caused by a decrease in the surface area of the inner mitochondrial membrane. The significance of these intra-mitochondrial changes to the rate-limiting step of steroidogenesis is discussed.

The contribution of the leak of protons across the mitochondrial inner membrane to standard metabolic rate

This paper presents and assesses the hypothesis that the proton leak across the mitochondrial inner membrane is an important contributor to standard metabolic rate, and that increases in the amount of mitochondrial inner membrane may be important in causing changes in proton leak and in the standard metabolic rate. The standard metabolic rate of an animal is known to be a function of body mass, phylogeny and thyroid status, and is largely attributed to the metabolically active internal organs. The total area of mitochondrial inner membrane in these organs correlates well with standard metabolic rate over a wide range of body masses in both ectotherms and endotherms. In hepatocytes isolated from rats, proton leak across the mitochondrial inner membrane accounts for about 30% of the resting oxygen consumption, and the distribution of control over respiration suggests that changes in mitochondrial inner membrane surface area will be accompanied by significant changes in the proton leak. This change in the leak will result in significant changes in resting oxygen consumption, but changes in ATP demand may also have a role to play in determining resting respiration rate. Extrapolation of these results to other tissues and other animals suggests that the hypothesis has the potential to explain a substantial proportion of the variation in standard metabolic rate with body mass, phylogeny and thyroid status. However, in most cases the quantitative contribution of proton leak compared to cellular ATP turnover has yet to be experimentally determined.

Capillary and mitochondrial unit in muscles of a large lizard.

We asked whether capillaries and mitochondria form a structural and functional unit in the musculature of the Cuban iguana (Cyclura nubila) similar to that found in mammals. We found a significant correlation between capillary length density [Jv(c, f)] and mitochondrial volume density [Vv(mt, f)] of the musculature with a slope that revealed that on average 3.5 km of capillaries were associated with each milliliter of mitochondria (vs. approximately 11 km/ml in mammals). These capillaries had a diameter of 9 microns (vs. 4.5 microns in mammals), and the mitochondria had a surface density of the inner membranes of 25 m2/ml (vs. 30-45 m2/ml in mammals). These dimensions resulted in ratios of capillary to mitochondrial volume (0.22 ml/ml) and capillary wall to mitochondrial membrane surface area (39 cm2/m2) that were similar in Cyclura to those found in mammals (approximately 0.18 ml/ml and 35-52 cm2/m2, respectively). Also in agreement with mammalian values were the average oxidative capacity of the mitochondria derived from maximum rate of O2 consumption (VO2max) during exercise at 37 degrees C and the inner mitochondrial membrane surface area [S(im)] of the musculature [VO2max/S(im) = 0.04 vs. 0.06-0.15 ml O2.m-2.min-1 in mammals]. These common structural and functional relationships support the notion that capillaries and mitochondria represent a similar fundamental unit in muscles of both Cyclura and mammals.

Quantitative morphologic changes in nephron structures and urinary enzyme activity pattern in sodium-maleate-induced renal injury.

The morphologic changes in sodium-maleate-induced acute renal injury in the rat were quantified by a stereologic analysis. The major changes were confined to an increase in endocytic vacuoles and a decrease in mitochondrial inner membrane surface area. These results were found to be linked to significantly increased urinary activities of the cytosolic of the cytosolic enzymes fructose-1,6-bisphosphatase (FBP) and lactate dehydrogenase, the lysosomal enzyme N-acetyl-beta-glucosaminidase (NAG) and the NAD-dependent mitochondrial isocitrate dehydrogenase (ICDH). The highest increase was found for NAG, followed by FBP and ICDH.

The relationship between renal metabolism and proximal tubule transport during ontogeny.

The proximal tubules of newborn and adult animals reabsorb a similar fraction of the filtered load of Na+ and H2O (65%-70%). In tubules from adult animals, transcellular, active Na+ reabsorption accounts for one-third of the total, while two-thirds occur passively through the paracellular pathway, driven by hydrostatic and oncotic forces (one-third) and by cell-generated effective osmotic and ionic gradients (one-third). Since two-thirds of the Na+ is reabsorbed passively and does not require energy, the mature proximal tubule has a high Na+/O2 molar ratio (48 Eq of Na+/mol of O2). Measurements of ouabain-sensitive oxygen consumption in suspensions of proximal tubules indicate that in newborn, aerobic metabolism can support about 50% of the net Na+ transport rate compared with the 33% in tubules from adult animals. Independent confirmation of the direct and proportional relationship between active Na+ transport and ouabain-sensitive O2 consumption exists for the adult but not for the newborn. However, measurements of epithelial conductances and of transepithelial hydrostatic and oncotic pressure differences indicate that passive paracellular fluxes can account for the remaining 50% of the proximal Na+ reabsorption in newborn. The high permeability of the proximal tubules of newborn animals to small molecular weight solutes suggests that cell-generated osmotic and ionic transepithelial gradients are minimal in the tubules of newborn animals. Yet in the newborn, the osmolality of the end proximal tubule fluid was found to exceed that in plasma. This indicates that osmotic gradients due to differences in reflection coefficients for preferentially reabsorbed solutes and Cl- do exist across the proximal tubules of the newborn and suggests that these gradients may contribute to Na+ and H2O reabsorption. If this is indeed the case, then the contribution of active and of hydrostatic and oncotic pressure-driven flows to the overall reabsorption of Na+ and fluid has been overestimated. Resolution of this discrepancy requires measurements of the reflection coefficients for HCO3- and Cl- in the proximal tubule of the newborn. The metabolic processes by which energy is supplied to renal proximal cells during development are also incompletely characterized. There is evidence that maturation of aerobic metabolism, Krebs cycle enzymes activity, and of the mitochondrial membrane surface area precede the development of net reabsorptive transport (Na+, H2O, HCO3, glucose). By contrast, maturation of Na(+)-K(+)-ATPase activity at the basolateral cell membrane follows that in reabsorptive transport and does not limit its development.(ABSTRACT TRUNCATED AT 400 WORDS)

Restoration effects of exogenous luteinizing hormone on the testicular steroidogenesis and Leydig cell ultrastructure.

In the present study, we explored the restoration effects of exogenous LH on Leydig cell ultrastructure and testicular steroidogenesis in rats that were deprived of endogenous LH via treatment with testosterone-17 beta-estradiol-filled Silastic implants for 10 days. Exogenous LH was supplied continuously via Alzet miniosmotic pumps at the rate of 1 microgram/h for 3, 6, 12, and 24 h or 1, 2, 4, 8, and 12 days. Testes were then perfused in vitro with medium containing 1) LH, 2) 20 alpha-hydroxycholesterol, or 3) pregnenolone substrate, which allowed us to assess LH-stimulated testosterone secretion, cholesterol side-chain cleavage activity, or the conversion of pregnenolone to testosterone, respectively. Other testes were perfusion fixed via the testicular artery for morphometric measurement of Leydig cell number and volume per testis and the surface area of Leydig cell cytoplasmic smooth endoplasmic reticulum (SER), inner mitochondrial membrane, and outer mitochondrial membrane. The results verified that Leydig cell smooth endoplasmic reticulum and inner and outer mitochondrial membrane surface areas are drastically diminished (P less than 0.05 vs. intact controls) by LH withdrawal. Also, the results verified that exogenous LH administered in situ restores Leydig cell ultrastructure and capacity to biosynthesize testosterone. However, the recovery of Leydig cell structure and steroidogenic reactions occurred at strikingly different rates upon restoration of LH after 10 days of the treatment with testosterone-17 beta-estradiol implants. For example, the restoration of testicular capacity to synthesize progesterone in response to LH stimulation or 20 alpha-hydroxycholesterol substrate was completed within 24 h. In contrast, the restoration of Leydig cell SER and testicular capacity to synthesize testosterone from pregnenolone was completed only after 8 days of continuous LH treatment (P greater than 0.05 vs. intact controls). Thus, our results show that LH rapidly restores Leydig cell post-LH receptor steroidogenic events up to and including cholesterol side-chain cleavage activity. Interestingly, there is no temporal association between the recovery of cholesterol side-chain cleavage activity and the surface area of inner mitochondrial membrane surface area. In contrast, 8 days are required to coincidentally restore SER surface area and the capacity of Leydig cells to synthesize testosterone from pregnenolone. We conclude that different cellular mechanisms are involved in the LH-dependent restoration of inner mitochondrial cholesterol side-chain cleavage activity and SER-associated conversion of pregnenolone to testosterone.