Human parathyroid hormone (1-34) increases urinary excretion of lysosomal enzymes in rats

The kidney is the major target of parathyroid hormone (PTH), and PTH influences the urinary excretion of calcium, phosphate and hydrogen ions. It was previously reported that the urinary, excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases after human PTH (hPTH) (1-34) infusion in normal subjects and idiopathic hypoparathyroidism patients, but not in pseudohypoparathyroidism type I patients. Here we report that intravenous infusion of hPTH(1-34) to rats transiently increased the urinary excretion of various lysosomal enzymes, such as beta-glucuronidase and acid phosphatase as well as NAG. However, it did not affect the urinary excretion of tubular brush border membrane enzymes, i.e. alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl transpeptidase. Human PTH(1-34) dose-dependently increased the urinary excretion of NAG in rats with a peak at 30 min, which returned to a baseline within 60 min. The increase in the urinary NAG excretion caused by hPTH(1-34) positively correlated with the increase in the urinary cAMP excretion (r = 0.844, p < 0.01), and infusion of dibutyryl cAMP at a dose of 20 mg/kg similarly increased the urinary excretion of NAG. These results suggested that the increase in the urinary excretion of lysosomal enzymes caused by hPTH(1-34) may be a functional response to hPTH(1-34) occurring in the renal tubules via PTH signaling pathway.     

Importance of urinary enzymes as a marker of post-ischemic proximal tubular involvement in rat

We have studied in rats the influence of renal ischemia on urinary excretion of three brush border membrane enzymes (gamma glutamyl transferase, alkaline phosphatase and leucine aminopeptidase) and of a lysosomal one (N-acetyl-B-D-glucosaminidase). Urines were collected over 24 hours periods during three days before and after a 45 minutes renal artery clamping. Urinary GGT, PAL and LAP excretion were significantly increased on the first day after renal ischemia, but returns to normal values on the second day. Urinary NAG activity increases on the first day, but contrary to the latter enzymes, reached to normal values only on the third day. Enzymuria seems to be a useful marker of tubular injury occurring after a temporary renal ischemia in the rat.     

Effect of synthetic 1-34 fragment of human parathyroid hormone on plasma adenosine 3',5'-monophosphate (cAMP) concentrations and the diagnostic criteria based on the plasma cAMP response in Ellsworth-Howard test

The effect of synthetic 1-34 fragment of human parathyroid hormone (hPTH(1-34] on plasma adenosine 3',5'-monophosphate (cAMP) in human subjects and the diagnostic criteria for the plasma cAMP response in an Ellsworth-Howard test were studied. 20 or 30 micrograms hPTH(1-34) and 200 USP Parathormone (Eli Lilly & Co.), infused intravenously over 5 min, produced very similar patterns of response in plasma cAMP, peak values being observed within 5 or 10 min after the end of the infusion. The maximum levels of plasma cAMP were over 111.5 pmol/ml in all of the normal subjects (n = 5) and patients with idiopathic hypoparathyroidism (n = 22), including those of children, but the plasma cAMP did not rise above 65.0 pmol/ml in pseudohypoparathyroidism (n = 7). There existed a significant correlation between the maximum plasma cAMP concentrations and increases in urinary cAMP excretion after infusions of both hPTH(1-34) and Parathormone. These results suggest that hPTH(1-34) has effects essentially identical to those of native PTH on plasma cAMP. We would like to propose a new diagnostic criterion in the Ellsworth-Howard test: a peak value of plasma cAMP over 100 pmol/ml after 30 micrograms hPTH(1-34) infusion is regarded as a normal response.     

The effect of cephaloridine on the stability of rat kidney lysosomes.

The administration of cephaloridine to rats caused a decrease in the excretion of acid phosphatase into the urine. The antibiotic itself had no effect on urinary acid phosphatase and inhibitors or proteolytic enzymes were not present in the urine from treated rats. Cephaloridine may therefore be stabilizing the lysosomal membrane in vivo and experiments with isolated lysosomes confirm this hypothesis. The lysosomal integrity was followed by measuring the acid phosphatase activity and the light scattering properties of the particles. A good correlation was obtained between these parameters in the case of thermal disruption and progesterone induced lysis of the lysosomes and low concentrations of cephaloridine (0.1-1.0 mmol/1) protected the lysosomes against this form of damage.     

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.     

Effects of a hepato-protective agent and a hepato-secreting chelator on cadmium-induced nephrotoxicity in Syrian hamsters

Cadmium (Cd)-induced nephropathy in male Syrian hamsters was treated with D/L-penicillamine (D/L-p) or neomynophagen C (NMC). The subcutaneous injection of CdCl₂, 3 mg/kg, three times a week led to marked renal damage, ie., increased proteinuria and the excretion of urinary N-acetyl-β-D-glucosaminidase (NAG) as compared with the saline-injected controls. Cd-treated hamsters that were injected intraperitoneally with D/L-p, 0.1 mg/kg, five times a week, showed less renal damage, including a reduction in urinary protein from 3.60±0.42 to 1.77±0.7 mg/d. NMC-treated hamsters showed a reduced excretion of NAG (from 1.47±0.34 to 0.91±0.68 u/d). The concentration of Cd in renal cortical tissue was reduced slightly (from 2.78±0.08 to 2.34±0.3 mg/g.prot) by NMC treatment, but not by D/L-p. The elevated malondialdehyde (MDA) in renal cortical tissue was unaffected by administering D/L-p or NMC. The concentration of glutathione (GSH) in the renal cortex was not elevated after administering Cd, but the ratio of the reduced to the oxidized GSH was elevated. The Cd induced liver dysfunction, as compared with untreated controls. The dysfunction was improved slightly by NMC administration, but not by that of D/L-p. Changes in renal morphology induced by Cd involving marked degeneration and necrosis of tubules as shown by light microscopy, were unaffected by treatment with D/L-p or NMC. We thus demonstrated the efficacy of D/L-p or NMC in treating the nephropathy induced by Cd in hamsters. The mechanism of therapeutic effect is not known.     

A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis

FTY720 has been originally developed as a new immunosuppressive agent, which prolongs graft survival in organ transplantation. Adrenomedullin (AM) participates in the regulation of sodium homeostasis and has renoprotective effects. The possible involvement of renal AM in the pathophysiology of glomerulonephritis (GN) and the effect of FTY720 has been evaluated in rats. HgCl2 (1 mg/kg body weight) was inoculated subcutaneously 3 times/week for a total of 2 weeks. FTY720 (3 or 10 mg/kg) was inoculated subcutaneously daily. The proteinuria, urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and serum total cholesterol levels were increased and serum albumin level was reduced in rats with HgCl2-induced GN compared with controls. FTY720 reduced proteinuria (3 mg/kg: -25%; 10 mg/kg: -41%), urinary NAG excretion (-11%; -52%) and total cholesterol level (-21%; -55%) in a dose-dependent manner. Renal AM level and its mRNA expression were increased in rats with GN compared with controls (Peptide Cortex: +69%; Medulla: +82%; mRNA Cortex: +25%). Interestingly, FTY720 additionally increased these levels (Peptide Cortex: +38%; Medulla: +39%; mRNA Cortex: +20%). Renal AM levels correlated with urinary NAG excretion and creatinine clearance. These results suggest that FTY720 suppresses the renal damage in rats with GN and renal AM may participate in the pathophysiology of GN and the renoprotective effects of FTY720.     

Dextran is resistant to lysosomal digestion in kidney tubules

Low molecular weight dextran (Rheomacrodex) was infused into dextran resistant rats in a dose of 5 g/kg body weight. The kidneys were studied by electron microscopy at different time intervals after infusion using a special fixative for the demonstration of dextran. The lysosomes of proximal tubule cells gradually accumulated dextran which remained in small amounts even after 10 days. In separate kidney slice experiments the ability of dextran-loaded proximal tubule lysosomes to digest absorbed proteins was determined using 125I-labelled lysozyme. There were no changes in lysosomal protein digestion. Labelled dextran was resistant to digestion in vitro by homogenates of rat or rabbit kidney cortex or isolated rat lysosomal enzymes. It is concluded that the protein absorption pathway and lysosomal protein catabolism is unchanged after tubular uptake of dextran despite pronounced ultrastructural alterations to the lysosomal system and that dextran is resistant to lysosomal digestion in renal proximal tubules.     

Anabolic effects of recombinant human parathyroid hormone (1 - 84) and synthetic human parathyroid hormone (1 - 34) on the mandibles of osteopenic ovariectomized rats with maxillary molar extraction.

In rodent osteoporosis models such as ovariectomized (OVX) rats, intermittently administered human parathyroid hormone (hPTH) has an anabolic effect in vertebrae and long bones. In the present experiments, subcutaneously injected hPTH(1 - 34) or hPTH(1 - 84) dose- and time-dependently increased bone mineral density (BMD) as measured by dual energy X-ray absorptiometry in mandibles, L2 to L4 vertebrae and femurs of such rats. The highest dose (15.9 nmol/kg, s. c.) of either peptide given four times weekly for 10 weeks completely reversed the effects of overiectomy on BMD. Significant elevation in lumbar BMD after 10 weeks was observed with hPTH(1 - 34) or hPTH(1 - 84) at 1.1 nmol/kg, whereas hPTH(1 - 34) at 1.1 and 4.2 nmol/kg significantly increased BMD of the whole bone and the metaphysis of the femur and the diaphysis of the bone, respectively. In contrast, significant effects of hPTH(1 - 84) administration on BMD increase in the femur were observed at 4.2 and 15.9 nmol/kg in the whole bone and the metaphysis, and in the diaphysis, respectively. Maxillary molar extraction left mandibular BMD in rats with intact ovaries unchanged, but significantly decreased mandibular BMD in OVX rats. Administration of hPTH(1 - 84) for 10 weeks in OVX rats without or with extraction significantly increased BMD in the mandibular molar region at doses of 15.9 and 4.2 nmol/kg, respectively, indicating that efficacy was increased by extraction. A significant BMD increase in the molar region in OVX rats with extraction occurred at only 1.1 nmol/kg of hPTH(1 - 34) and 4.2 nmol/kg of hPTH(1 - 84). Also, BMD of the ramus region was increased by administration of both peptides to a lesser extent than that of the molar region in these rats. Thus, intermittent administration of hPTH, especially hPTH(1 - 34), has an anabolic effect on bone, particularly alveolar bone. Such treatment may increase alveolar bone mass in postmenopausal women with osteoporosis.     

Latency of acid hydrolases in rat kidney cortex

1. Some lysosomal populations in the rat kidney cortex appear to be mechanically weak and are readily disrupted by gentle homogenization, while other populations remain intact even after repeated homogenization. 2. Lysosomes in the rat kidney cortex appear to be resistant to hypertonic media but are readily disrupted under hypotonic conditions. 3. Lysosomes in rat kidney cortex are readily disrupted when incubated in isotonic sucrose at 37 degrees C. 4. Measurement of total and free activity of three acid hydrolases: N-acetyl-beta-D-glucosaminidase (NAG), acid beta-galactosidase and acid beta-glycerophosphatase, indicates that the latency of these enzymes is relatively low in the homogenate (10-29%) and the ML-fraction (14-42%), but high (60-95%) in the purified large lysosomes (protein droplets). 5. The latency of purified small lysosomes is relatively lower (30-60%) than that of large lysosomes, suggesting that small lysosome populations are relatively permeable to the acid hydrolase substrates. 6. Latency variations of acid hydrolases amongst subcellular fractions appear to reflect the heterogeneity of lysosomal populations present in the kidney cortical homogenate.     

Human parathyroid hormone related protein fragment-(1-34) had glucose-6-phosphate dehydrogenase activity on distal convoluted tubules in cytochemical bioassay

In the present study, the action of parathyroid hormone related protein (PTHrP) on glucose-6-phosphate dehydrogenase (G6PD) activity of the distal convoluted tubules was examined utilizing cytochemical bioassay (CBA). Recently full amino acid residues of human PTHrP (hPTHrP), one of the causative agents of HHM, was identified based on the cDNA clone using BEN cells. We synthesized hPTHrP-(1-34) and examined the effect of this protein on G6PD activities on the distal convoluted tubules, and compared its bioactivity to that of human parathyroid hormone (hPTH)-(1-84). hPTHrP-(1-34) stimulated G6PD activity in a log linear fashion with equivalent activity to that of hPTH-(1-84) on a molar basis. Conclusively, we found that PTHrP act on distal convoluted tubules similar to hPTH.     

The electron density of light and dark lysosomes in the proximal convoluted tubule of the rat kidney

The present paper deals with the electron density of the lysosomal matrices in the renal proximal-convoluted-tubule cells of Wistar rats (20-60 days of age). Its purpose was to determine which physicochemical factors influence the electron density of lysosomes, and how their electron density is affected by various methods of fixation.--3 types of lysosomes can be distinguished in the proximal-convoluted-tubule cell; namely light, intermediate and dark lysosomes, which have an electron density lower, equal to or higher than the surrounding cytoplasm. All 3 types of lysosomes were invariably present after all methods of fixation tested. Light, intermediate and dark lysosomes differ in several respects. Staining intensity with uranyl acetate, lead citrate and potassium permanganate, osmiophilia, basophilia (in semithin sections) and --probably most importantly--the physical mass density of the lysosomal matrix are all low in light lysosomes, higher in intermediate lysosomes and highest in dark lysosomes. Light, intermediate and dark lysosomes of the proximal convoluted tubule do not form discrete classes, but one continuous spectrum of lysosomes of increasing electron density.     

Antineoplastic therapy and urinary enzymes. Preliminary note on the determination of urinary N-acetyl-beta-D-glucosaminidase in patients treated with cisplatin

We discussed the diagnostic value of urinary enzymes as non invasive test of renal integrity in medicine. Then we assayed the N-Acetyl-beta-D-glucosaminidase (NAG) excretion in urines of inpatients receiving cis-platinum, a useful anti-cancer but nephrotoxic agent fort the proximal tubule; we used the colorimetric instead of fluorimetric procedure, recently described in detail by Price who tested it on patients with a variety of renal diseases. The results demonstrated the sensibility and the specificity of colorimetric procedure, and therefore we concluded that the assay of urinary NAG activity may be used in the clinical chemistry and toxicology laboratory, because the test is simple to perform.     

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.     

Effects of oestradiol, testosterone and medroxyprogesterone on subcellular fraction marker enzyme activities from rat liver and brain

The following enzymes have been studied (subcellular fractions are shown between parentheses): NAG and beta-glucuronidase (lysosomes); SDH (mitochondrial); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and (Na+, K+)Mg2+ ATPase (plasma membranes). Alterations on their activities were observed after subcutaneous injection of sex hormones, compared with controls. NAG activity from liver was always significantly decreased in lysosomal and microsomal fractions after the hormonal treatment. In the same conditions, NAG from brain was always increased. beta-Glucuronidase behaves like NAG in brain; in liver it was not modified by testosterone and it was slightly increased in lysosomal fraction after oestradiol treatment. SDH activity was not modified in mitochondrial fractions from liver, but this activity was always significantly increased in brain. Glucose-6-phosphatase activity was always significantly decreased in microsomal fractions from liver. It was increased in brain after oestradiol and testosterone injection, but medroxyprogesterone treatment caused a decreased activity. 5'-Nucleotidase and (Na+, K+)Mg2+ ATPase from brain were significantly increased in microsomal fractions by oestradiol and testosterone. Medroxyprogesterone, however, caused an increase in ATPase, but did not affect 5'-nucleotidase. Both activities in liver were decreased by oestradiol and increased by testosterone, but medroxyprogesterone caused (Na+, K+)Mg2+ ATPase to rise and 5'-nucleotidase to fall.     

CISPLATIN-INDUCED VOMITING DEPENDS ON CIRCADIAN TIMING

We examined whether the clock time of cisplatin plus antiemetic and diuretic administration affects the amount of cisplatin-associated emesis and severity of renal toxicity. We treated 22 patients with urogenital cancer with two courses of chemotherapy containing 70 mg/m² of cisplatin. Cisplatin together with furosemide was administered in the morning (05:00) or evening (17:00) during two courses 1 month apart in a crossover fashion. Ondansetron was given either before or after cisplatin to control nausea and vomiting. The number of vomiting episodes, serum creatinine, serum urea nitrogen (BUN), creatinine clearance, and urinary β-N-acetyl glucosamidase (NAG) concentration were evaluated before and after each treatment course. Regardless of the timing of ondansetron, morning compared to evening cisplatin was always associated with greater vomiting in the first treatment course. However, prophylactic administration of ondansetron markedly diminished the impact of the clock time of cisplatin administration. Serum creatinine transiently decreased rather than increased 14 days after cisplatin and furosemide administration, while NAG excretion increased 3 days after cisplatin and furosemide administration. In the first course, serum creatinine levels were similar regardless of the clock time of cisplatin and furosemide administration. However, in the second course, serum creatinine rose in patients given evening cisplatin and furosemide, while it remained unchanged in those given morning cisplatin and furosemide. Moreover, the first course morning cisplatin and furosemide treatment was associated with less change in NAG excretion (less kidney toxicity) than the first course of evening cisplatin and furosemide treatment. The second course evening cisplatin and furosemide treatment was associated with an increase in NAG excretion compared to the first course of treatment, while morning cisplatin and furosemide treatment in the second course showed less change in NAG excretion compared to the first course. The clock time of cisplatin administration had an impact on the frequency of emesis. Prophylactic ondansetron, however, diminished the time-of-day dependency of cisplatin-induced vomiting. Administration of cisplatin and furosemide in the morning rather than evening appears to cause less renal damage, and this damage may be further reduced with aggressive hydration and routine administration of furosemide. (Chronobiology International, 18(5), 851-863, 2001)     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Bioactivity of amino terminal fragments of parathyroid hormone and parathyroid hormone related peptide in rat kidney slices: no effect of dietary phosphate deprivation

Humoral hypercalcemia of malignancy has been associated with the production of a recently cloned peptide human parathyroid hormone related protein (hPTHRP). One of the markers of this disease is an increased urinary excretion of cyclic AMP. The postreceptor mechanism of action and physiological role of hPTHRP remain obscure. To study the activity of hPTHRP 1-34 compared to rat and human parathyroid hormone (PTH) 1-34 we incubated these peptides with rat kidney slices and measured the cyclic AMP generated in the supernatant. hPTHRP 1-34 was equipotent with human PTH 1-34 but both were 5 times less active than rat PTH 1-34. Previous studies have suggested that a low dietary phosphate intake results in renal resistance to the phosphaturic action of PTH perhaps mediated by reduced adenylate cyclase activation by PTH. To determine whether, during dietary phosphate restriction, hPTHRP 1-34 has actions different from hPTH 1-34 we studied their effects following dietary phosphate deprivation. Dietary phosphate restriction had no significant effect on the cyclic AMP generating activity of any of the peptides. We conclude that hPTHRP 1-34 may be operating through similar mechanisms as human PTH 1-34 and that the previously observed effects of dietary phosphate deprivation on PTH mediated cyclic AMP generation in a broken cell preparation do not occur in intact cell preparations.     

In vivo endocytosis by bristle coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. II. The endosomal-lysosomal transformation

The events leading to lysosomal activity in the sinus endothelium of the rat liver have been studied by means of intravascularly injected ferritin at time intervals ranging from 0.5 min to 1 hr after administration. From 6 min on, the dense body-type lysosomes contain ferritin. There are direct luminal communications of transfer tubules with these lysosomes. In time, there is a marked progressive increase in the number of ferritin-containing dense body-type lysosomes. No formation of lysosomes de novo nor a direct fusion of endosomes with lysosomes has been observed. Endosomes, however, continue to be formed as endocytosis continues. These observations are interpreted as indicating a transport of hydrolytic enzymes by the transfer tubules to the newly formed ferritin containing endosomes, which in this way are transformed into ferritin containing lysosomes. The ferritin-containing lysosomes increase considerably in size by fusing with each other. Continued endocytosis of ferritin leads to an increase of ferritin density in the dense bodies. This increase in particle density cannot be explained solely on the basis of transport by luminal fusion of the endocytic organelles, but requires an active transport mechanism. Administration of low doses of ferritin shows that the bristle coated pits of the sinus endothelium have a high degree of in vivo affinity for protein and that this endothelium must be considered to be an avid catabolic endocytic system.