High-calcium diet modulates effects of long-term prolactin exposure on the cortical bone calcium content in ovariectomized rats

High physiological prolactin induced positive calcium balance by stimulating intestinal calcium absorption, reducing renal calcium excretion, and increasing bone calcium deposition in female rats. Although prolactin-induced increase in trabecular bone calcium deposition was absent after ovariectomy, its effects on cortical bones were still controversial. The present investigation, therefore, aimed to study the effect of in vivo long-term high physiological prolactin induced by either anterior pituitary (AP) transplantation or 2.5 mg/kg prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of normal adult rats has not been reported, we first determined mRNA expression of both short- and long-form PRLRs at the cortical sites (tibia and femur) and trabecular sites (calvaria and vertebrae) by using the RT-PCR. Our results showed the mRNA expression of both PRLR isoforms with predominant long form at all sites. However, high prolactin levels induced by AP transplantation in normal rats did not have any effect on the femoral bone mineral density or bone mineral content. By using (45)Ca kinetic study, 2.5 mg/kg prolactin did not alter bone formation, bone resorption, calcium deposition, and total calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical total calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that 2.0% wt/wt high-calcium diet significantly increased the tibial total calcium content in 9-wk-old young AP-grafted ovariectomized rats but decreased the tibial total calcium content in 22-wk-old adult rats. As for the vertebrae, the total calcium contents in both young and adult rats were not changed by high-calcium diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.     

Effects of promethazine HCl on osteoporotic femora of adult castrated male rats

Promethazine HCl (Phenergan) was added to the drinking water (2.4-9.6 mg/kg/day) of castrated adult male Holtzman rats. One group of castrates and a group of normal rats received no drug and served as controls. Treatment was started at castration and continued for 4 months. After controlling for body weight, analyses of covariance and Student-Newman-Keuls tests revealed that promethazine did not prevent the development of femoral osteoporosis in the castrate rats. However, control rats (normal and untreated castrates) had significantly wider femora and thicker cortices at midshaft than did promethazine-treated animals. The results indicated that promethazine HCl had no effect on the development of femoral osteoporosis and retarded normal femoral expansion in the adult castrate male rats.     

Effects of male rat urine on norepinephrine levels in the accessory olfactory bulb of young and aged female rats.

Norepinephrine (NE) concentrations in the accessory olfactory bulbs (AOB) of young (4-5 months) and old (25-26 months) ovariectomized Sprague-Dawley rats, which were implanted with a 17 beta-estradiol silastic capsule and then exposed to male rat urine, were studied. The unilateral vomeronasal organ was removed in all rats one week before exposure to the urine stimulation. The NE level in the AOB of this surgical side served as the control. Urine collected from young adult male rats was poured into the female's cage at 12:00h and the animals were sacrificed before and 1, 2, or 3 hours after the male urine was given. The NE basal levels in the AOB of young rats decreased significantly at 14:00h compared to those at 12:00h, while no obvious changes in the NE concentrations were observed in the AOB of old rats during the same period. Two hours after continuous exposure to male urine, the NE concentrations in the AOB of young rats markedly increased, but no similar response occurred in the old rats. These data suggest that the noradrenergic functions in the AOB under basal conditions as well as in response to pheromonal stimulation may be modified with increasing age.     

MyoD and myogenin protein expression in skeletal muscles of senile rats

We analyzed the level of protein expression of two myogenic regulatory factors (MRFs), MyoD and myogenin, in senile skeletal muscles and determined the cellular source of their production in young adult (4 months old), old (24, 26, and 28 months old), and senile (32 months old) male rats. Immunoblotting demonstrated levels of myogenin approximately 3.2, approximately 4.0, and approximately 5.5 times higher in gastrocnemius muscles of 24-, 26-, and 32-month-old animals, respectively, than in those of young adult rats. Anti-MyoD antibody recognized two major areas of immunoreactivity in Western blots: a single MyoD-specific band (approximately 43-45 kDa) and a double (or triple) MyoD-like band (approximately 55-65 kDa). Whereas the level of MyoD-specific protein in the 43- to 45-kDa band remained relatively unchanged during aging compared with that of young adult rats, the total level of MyoD-like immunoreactivity within the 55- to 65-kDa bands was approximately 3.4, approximately 4.7, approximately 9.1, and approximately 11.7 times higher in muscles of 24-, 26-, 28-, and 32-month-old rats, respectively. The pattern of MRF protein expression in intact senile muscles was similar to that recorded in young adult denervated muscles. Ultrastructural analysis of extensor digitorum longus muscle from senile rats showed that, occasionally, the area of the nerve-muscle junction was partially or completely devoid of axons, and satellite cells with the features of activated cells were found on the surface of living fibers. Immunohistochemistry detected accumulated MyoD and myogenin proteins in the nuclei of both fibers and satellite cells in 32-month-old muscles. We suggest that the up-regulated production of MyoD and myogenin proteins in the nuclei of both fibers and satellite cells could account for the high level of MRF expression in muscles of senile rats.     

The effects of dichloromethylene biphosphonate on osteoporotic femora of adult castrate male rats

In a previous study we reported that castration of adult male rats resulted in femoral osteoporosis 4 months later. The present study tested the effects of dichloromethylene biphosphonate (Cl2MBP, formerly Cl2MDP), a bone resorption inhibitor, on the development of osteoporosis in femora of adult castrated male rats. One-year-old male Holtzman rats were assigned to three groups: sham-operated controls; castrated-no treatment; castrated + Cl2MBP injections 3 times/week. The animals were sacrificed 4 months later. Femora from untreated castrated rats were osteoporotic, confirming our earlier study. Femora from Cl2MBP-treated castrates did not differ from those of controls and were significantly denser and more robust than those of untreated castrated rats. The results indicated that Cl2MBP treatment started immediately after castration prevented the development of femoral osteoporosis in adult castrated male rats.     

bFGF increases collateral blood flow in aged rats with femoral artery ligation

We tested the hypothesis that aged animals are as responsive as the young adult animals in expanding collateral vasculature under a similar treatment of basic fibroblast growth factor (bFGF). Two age groups of male Fischer 344 rats (11 mo old; n = 32, 23 mo old; n = 43) weighing approximately 385 g were subdivided into normal, acute ligation [femoral artery (FA) ligated 3 days before blood flow (BF) measurement] or ligated groups for 16 days and received recombinant human bFGF intra-arterial infusion at doses of 0, 0.5, 5, and 50 microg x kg(-1) x day(-1). BF was determined with (85)Sr- and (141)Ce-labeled microspheres during treadmill running at 15 and 20 m/min at 15% grade. Blood pressure (BP) values were approximately 149 and approximately 163 mmHg (p < 0.05); heart rates were approximately 496 and approximately 512 beats/min in the aged and young adult groups during running, respectively. Maximal collateral BF values were confirmed by no additional BF increase in the calf muscle at the higher speed. Ligation of the FA for 3 days reduced the BF reserve to the calf muscle by approximately 90%. Calf muscle BF was modestly greater (10 ml x min(-1) x 100 g(-1)) by 16 days in the carrier group. bFGF infusion expanded collateral BF in a dose-dependent manner with an increase of 33 and 42 ml x min(-1) x 100 g(-1) (P < 0.001) in the 5 and 50 microg x kg(-1) x day(-1) bFGF groups, respectively. Aged animals showed similar BF improvements as observed with the adult groups in response to ligation surgery and bFGF treatment. Our data indicate that the aged rats (approximately 23 mo old) remain responsive to exogenous bFGF induced in developing collateral-dependent BF as the young adult (approximately 11 mo old) controls. This suggests that the influence of bFGF in expanding collateral BF should not be preempted in the aged group, the population most affected by peripheral arterial insufficiency.     

Influence of clenbuterol on bone metabolism in exercised or sedentary rats.

This paper reports that the selective beta(2)-adrenergic receptor agonist clenbuterol affects bone metabolism in growing 3-mo-old male Wistar rats treated over 8 wk. Thirty-two 3-mo-old growing Wistar rats weighing 234 +/- 2 g were assigned to a progressive isometric force, strength-training exercise program plus oral clenbuterol (2 mg x kg body wt(-1) x day(-1)) for 5 days each week, exercise program without clenbuterol 5 days each week, no exercise program plus oral clenbuterol (2 mg x kg(-1) x day(-1)) for 5 days each week, or no exercise without clenbuterol 5 days each week. At the end of 8 wk, lean mass, fat mass, and right total femoral, distal metaphyseal femoral, and diaphyseal femoral bone mineral density were measured by Hologic QDR 4,500 dual X-ray absorptiometry (DEXA) technique. Left femoral bones were harvested after death on day 58, and femoral resistance was determined by three-point bending testing. We found that fat mass was decreased in rats given strength training exercise and decreased further in rats treated with clenbuterol. Lean mass was increased in clenbuterol-treated animals. Strength-training exercise appeared to have no effect on bone mineral density, serum osteocalcin, or urinary deoxypyridinoline. However, clenbuterol treatment decreased femoral length, diameter, bone mineral density, and mechanical resistance. Clenbuterol had no effect on osteocalcin but increased urinary deoxypyridinoline. We concluded that clenbuterol treatment decreased bone mineral density and increased bone resorption independent of the level of exercise rats were given.     

Age-related differences in synaptic plasticity following muscle unloading

The objective of the present investigation was to determine the effects of muscle unloading-a form of subtotal disuse- on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n=8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT-4 expression. Quantitative analyses revealed that aged controls displayed significant (p<0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls>young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT-4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus.     

Dichloromethylene biphosphonate retards femoral expansion in normal and castrated adult male rats

This study reports the effects of dichloromethylene biphosphonate (Cl2MBP), an inhibitor of bone remodeling, on femoral expansion in four groups of adult male rats; (1) sham-operated controls; (2) sham operated + injections of Cl2MBP; (3) castrated (osteoporotic), and (4) castrated + injections of Cl2MBP. After controlling for body weight, analyses of covariance revealed significant differences in total femoral width between animals that had received Cl2MBP and those that had not. The results indicated that Cl2MBP treatment retarded femoral expansion in both castrated and normal adult male rats.     

Norepinephrine levels in the vomeronasal system and their responses to male urine in young and aged female rats

Norepinephrine (NE) levels in brain areas of the vomeronasal system in young (4-5 months) and aged (25-26 months) ovariectomized Sprague-Dawley rats, which were implanted with a 17 beta-estradiol silastic capsule and then exposed to male rat urine, were investigated. The unilateral vomeronasal organ was removed in all rats one week before exposure to urine stimulation. NE levels in the medial nucleus of the amygdala (MA), medial preoptic area (MPOA), ventromedial nucleus of hypothalamus (VMH) and bed nucleus of stria terminalis (BST) were measured. NE concentrations in these brain areas of the surgical side served as the control. Urine collected from young adult male rats was poured into the female's cage at 12:00h and the animals were sacrificed before and 1, 2, or 3 hours after the male urine was given. The NE basal levels in the MA and MPOA of young rats decreased significantly from 13:00h to 15:00h, and those in young rat VMH declined markedly from 13:00h to 14:00h compared to those at 12:00h. No marked alterations in NE basal levels in young rat BST were found. In contrast, no obvious changes in the NE concentrations were observed in these brain areas of old rats. Continuous exposure to male urine did not affect the NE levels in any of these brain areas of young and aged rats. We concluded that (1) the time-dependent fluctuation of the NE basal levels in some brain areas of the vomeronasal system in female rats is age-related, and (2) the NE in all these nuclei of the vomeronasal system is not involved in pheromone-induced effects.     

Age-related changes in calcium and phosphorus uptake by rat small intestine

The purpose of these experiments was to determine whether there are changes in intestinal Ca and P uptake with age and whether the regulation of Ca and P uptake changes with age. Experiments were performed in male Fischer 344 rats aged 2-3 months (young), 12-14 months (adult) and 22-24 months (old). Ca and P uptake were measured simultaneously by incubating everted intestinal sacs in a buffered salt solution containing radiolabeled 0.25 mM Ca and 1.0 mM P for 15 min. Ca uptake declined by over 50% with age in the duodenum, and P uptake showed a similar decline in both the duodenum and jejunum. The biggest decrease was seen between the young and adult age groups. These decreases in uptake were paralleled by decreases in serum 1,25-dihydroxyvitamin D with age. Administration of 1,25-dihydroxyvitamin D-3 increased Ca uptake by 50-65% in the duodenum and increased P uptake by 85-120% in the duodenum and jejunum of both young and adult rats. Although 1,25-dihydroxyvitamin D-3 increased uptake by about the same percentage in each age group, the maximal uptake was much greater in the young than in the adult. Feeding a low-Ca diet increased duodenal Ca uptake by 68% and increased serum 1,25-dihydroxyvitamin D over 2-fold in young rats. There was no significant increase in either parameter in adult rats fed a low-Ca diet. However, duodenal P uptake was stimulated by a low-Ca diet by 87% in young rats and by 51% in adult rats. These results demonstrate that there is an age-related decline in Ca and P uptake by the intestinal mucosa. In addition, there is decreased capacity of 1,25-dihydroxyvitamin D-3 and a low-Ca diet to stimulate intestinal uptake in the adult.     

Vagal afferent activity and body temperature in 3 to 10-day-old and adult rats

This study examined the possible contribution of vagal stretch receptor activity to the increased power of the Hering-Breuer reflex in hyperthermia in rats during the early postnatal period. Experiments were performed on 10 anesthetized (pentobarbital 40 mg/kg, i.p.) 3 to 10-day-old (body weight of 16 +/- 1 g; SE) and, for comparison, 18 adult Sprague-Dawley rats (body weight of 336 +/- 35 g). Animals were tracheostomized and artificially ventilated with oxygen. The left vagus nerve was cut. In adult animals, single receptor fibers or a bundle of a few fibers were recorded using a bipolar stainless-steel electrode under mineral oil. In the young rats, a suction electrode filled with normal saline was used. Positive pressure of either 5 or 10 cmH2O was applied to the trachea when the respirator was turned off. The vagal activity was amplified and monitored on a storage oscilloscope for calculation of the frequency of vagal afferent activity during a given pressure application at different rectal temperatures (T(R); range 28 to 42 degrees C). In total, 30 and 31 sets of vagal activity in the young and adult rats, respectively, were analyzed. In all cases, an increase in tracheal pressure (P(TR)) from 5 to 10 cmH2O increased the frequency of vagal firing. The increase was greater in the adult versus the young animals; at 36 degrees C the increase was 49 +/- 11% and 16 +/- 3% in the adult and young rats, respectively (P < 0.01). In all animals, vagal receptors showed temperature-sensitivity, but less so in the young than in the adult rats (P < 0.0004 and P < 0.003; for P(TR) of 5 and 10 cmH2O, respectively). In addition, the relationship between temperature-sensitivity and T(R) had significant slopes (P < 0.001 for both inflation pressures) in the adults but not in the young rats, indicating that in the latter the temperature-sensitivity of vagal receptors is independent of TR. These results imply that temperature-sensitivity of vagal receptors could have contributed to the increased power of the Hering-Breuer reflex in rats during the early postnatal period in the warmer environment.     

Adaptive responses of 25-hydroxyvitamin D3 1-alpha hydroxylase expression to dietary phosphate restriction in young and adult rats

Regulation of vitamin D metabolism alters with age. The present study is undertaken to investigate if the loss of renal 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) production in response to dietary phosphate (P) restriction in adult rats is due to an alteration in the renal expression of 25-hydroxyvitamin D(3) 1-alpha hydroxylase (1-OHase). Young (4-6 weeks old) and adult (12-14 weeks old) male Sprague Dawley rats were fed either normal P (NPD) or low P diet (LPD) for 0-5 days. Basal expression of 1-OHase protein was higher in adult rats. Young rats, but not adult rats, significantly increased 1-OHase protein and mRNA expressions in response to LPD in a time-dependent manner. To determine if the stability of renal 1-OHase protein changes with LPD feeding, young and adult rats fed either NPD or LPD for 5 days were injected intravenously with cycloheximide (CHX), a protein synthesis inhibitor. CHX decreased 1-OHase protein expression in young rats fed NPD. However, CHX did not alter 1-OHase protein expression in young rats fed LPD nor in adult rats fed either diet. The results indicate that the stability of renal 1-OHase protein increased with age and that LPD increased its stability only in young rats.     

Time course changes to structural,mechanical and material properties of bone in rats after complete spinal cord injury

Objective:Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats.Methods:8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy.Results:The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone.Conclusion:Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.     

Vascular damage after fractionated whole-brain irradiation in rats

Whole-brain irradiation of animals and humans has been reported to lead to late delayed structural (vascular damage, demyelination, white matter necrosis) and functional (cognitive impairment) alterations. However, most of the experimental data on late delayed radiation-induced brain injury have been generated with large single doses or short fractionation schemes that may provide a less accurate indication of the events that occur after clinical whole-brain radiotherapy. The pilot study reported here investigates cerebral vascular pathology in male Fischer 344 rats after whole-brain irradiation with a fractionated total dose of 137Cs gamma rays that is expected to be biologically similar to that given to brain tumor patients. The brains of young adult rats (4 months old) were irradiated with a total dose of 40 Gy, given as eight 5-Gy fractions twice per week for 4 weeks. Brain capillary and arteriole pathology was studied using an alkaline phosphatase enzyme histochemistry method; vessel density and length were quantified using a stereology method with computerized image processing and analysis. Vessel density and length were unchanged 24 h after the last dose, but at 10 weeks postirradiation, both were substantially decreased. After 20 weeks, the rate of decline in the vessel density and length in irradiated rats was similar to that in unirradiated age-matched controls. No gross gliosis or demyelination was observed 12 months postirradiation using conventional histopathology techniques. We suggest that the early (10-week) and persistent vascular damage that occurs after a prolonged whole-brain irradiation fractionation scheme may play an important role in the development of late delayed radiation-induced brain injury.     

Bones benefits gained by jump training are preserved after detraining in young and adult rats.

We investigated the osteogenic responses to jump training and subsequent detraining in young and adult male rats to test the following hypotheses: 1) jump training has skeletal benefits; 2) these skeletal benefits are preserved with subsequent detraining throughout bone morphometric changes; and 3) there are no differences between young and adult rats during detraining in terms of the maintenance of exercise-induced changes. Twelve-week-old (young) and 44-wk-old (adult) rats were divided into the following four groups: young-sedentary, young-exercised, adult-sedentary, and adult-exercised. The exercised groups performed jump training (height = 40 cm, 10 jumps/day, 5 days/wk) for 8 wk followed by 24 wk of being sedentary. Tibial bone mineral content and bone mineral density in vivo significantly increased with jump training, and the effects were maintained after detraining in both the young and adult exercised groups, although the benefits of training became somewhat diminished. After 24 wk of detraining, the beneficial effects of training on bone mass and strength were preserved and associated with morphometric changes, such as periosteal perimeter, cortical area, and moment of inertia. There were no significant age-exercise interactions in such parameters, except for the periosteal perimeter. These results suggest that there are few differences in bone accommodation and maintenance by training and detraining between young and adult rats.     

Treadmill running starting 3 months after orchidectomy restores femoral bone mass in rats

The present study was designed to provide data on the effects on femoral bone of endurance training starting only 3 months after orchidectomy in rats. A total of 70 Wistar male rats were used at 8 weeks of age. On day 0 of the experiment, 10 rats were killed by cervical dislocation to be used as first controls. Among the 60 other animals, half was surgically castrated (CX) or sham operated (SH). On day 90, 10 CX and 10 SH were killed and used as intermediary controls (ICX and ISH). Among the other 20 CX and 20 SH, 10 within each group (CXE, SHE) were selected for treadmill running (60% maximal oxygen uptake, 1 h x day(-1), 5 days x week(-1) for 12 weeks). The 20 other rats were used as sedentary controls (CXR, SHR) and killed (as runners) on day 180. On day 90 femoral bone density (BMD) and mineral content (BMC) were lower in ICX than in ISH. On day 180 total femoral BMD was lower in CXR than in CXE. Simultaneously metaphyseal femoral BMD was lower in CXR than in CXE, SHR or SHE. Furthermore, at that time, no significant difference concerning BMD and BMC was observed between SHR and CXE. This would indicate that treadmill running starting only 3 months after orchidectomy is able to restore BMD and BMC to control values, mainly by inhibiting bone resorption (as shown by decreased urinary deoxypyridinoline excretion in CXE) without decreasing osteoblastic activity (evaluated by plasma osteocalcin concentration).     

Age‐related differences in synaptic plasticity following muscle unloading

The objective of the present investigation was to determine the effects of muscle unloading—a form of subtotal disuse— on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n = 8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT‐4 expression. Quantitative analyses revealed that aged controls displayed significant (p < 0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls > young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT‐4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 246–256, 2003     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Metabolic Response to Renal Compensatory Growth

Forty-eight hours after unilateral nephrectomy in young male Sprague-Dawley rats the concentrations of free methionine, alanine and tyrosine in renal cortical tissue were increased by 15-65 percent while the corresponding plasma concentrations decreased by 23-35 percent. The renal cortical concentrations of valine and leucine increased by 41 percent and 26 percent while plasma concentrations remained unchanged. The cortical concentrations of ornithine, serine and threonine remained unchanged while the plasma concentration decreased by approximately one-third. The total free amino acid contained in the cortex was not changed, while total free amino acids in plasma decreased by 7 percent. These data are thought to reflect an increased uptake of methionine and tyrosine into renal cells during compensatory hypertrophy, and an increased incorporation into renal protein of serine, threonine and ornithine. All these changes as well as all other biochemical changes accompanying compensatory hypertrophy with the exception of an increase of the RNA/DNA ratio were prevented by starvation for 48 hours after unilateral nephrectomy.In young male Sprague-Dawley rats and adult male Charles River mice, the incorporation of ¹⁴C-choline into acid-insoluble phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) was already accelerated 5 minutes after contralateral nephrectomy and further rose to +68 ± 7 percent within 20 minutes to 3 hours. Incorporation of ¹⁴C-choline into phospholipids remained accelerated for two to three days and reflected increased rates of phospholipid synthesis rather than increased choline uptake. Three hours after unilateral nephrectomy in mice, incorporation of i.p. injected ¹⁴C-choline into phospholipids was accelerated 25 percent. The rate of turnover of free labelled renal phospholipids was not accelerated during compensatory renal growth. The very early increase of choline incorporation into phospholipids after contralateral nephrectomy, therefore, appears to reflect an increased rate of synthesis of membrane material.     

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16-day-old rats and from cortical regions and brainstem, respectively, of 40-day-old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16-day-old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16-day-old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one-third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16-day-old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16-day-old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40-day-old rats was 4-fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16-day-old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin-related membranes, although the heavy fraction from 16-day-old rats also may be substantially contaminated with membranes which are unrelated to myelin.