Search

Photomicrograph of a longitudinal section taken in a lingual-buccal plane through the entoflexid region of a permanent mandibular left third premolar (307) tooth of a 4-year-old horse showing cementum covering the whole tooth apart from the occlusal surface. A thin layer covers the root (Re). The slightly thicker but uniform alveolar cementum (Ac) covers the alveolar crown. In the gingival region, the peripheral cementum is much thicker (Gc). The cementum forms a tight junction with the enamel folds. Cementum of the entoflexid can be identified in the section ( * ). Dentin (D) juxtaposed to the pulp (P) extends to the occlusal surface. Secondary dentin (Sd) can be identified on the buccal aspect. The tooth is supported in the alveolar bone (Ab) by the periodontal ligament (Pdl) [original magnification x 1.7]. [ABSTRACT FROM AUTHOR]

Morphological and functional changes in endothelial and interstitial cells are considered central to myxomatous degeneration of the canine mitral valve (endocardiosis). The aim of this study was to describe and quantify changes in valve endothelial cells (VECs), interstitial cells (VICs) and the extra-cellular matrix (ECM) of the sub-endothelial zone of diseased valves using a combination of transmission electron microscopy, stereology and computer-aided image analysis. Marked degradation of the endothelium was evident in diseased valves, which coincided with significant degradation of the local ECM (P < 0.001). There were decreases and increases in the numbers of VECs and VICs, respectively, in diseased valves, with particular accumulation of VICs subjacent to the valve surface (P < 0.01). Overall, VICs were more pleomorphic than VECs in both normal and diseased valves, but for VECs, the degree of pleomorphism was significantly different in diseased valves (P < 0.0001). The findings of the study confirm that canine myxomatous mitral valve disease is associated with marked endothelial damage, with attendant proliferation of subjacent activated myofibroblasts. The fact that similar endothelial changes are present in normal valves suggests these processes not only contribute to valve pathology, but may also represent life-long valve remodelling. [ABSTRACT FROM AUTHOR]

Ten normal cheek teeth (CT) were extracted at post mortem from donkeys that died or were euthanased for humane reasons. Decalcified histology was performed on three sections (sub-occlusal, mid-tooth and pre-apical) of each tooth, and undecalcified histology undertaken on sub-occlusal sections of the same teeth. The normal histological anatomy of primary, regular and irregular secondary dentine was found to be similar to that of the horse, with no tertiary dentine present. Undecalcified histology demonstrated the normal enamel histology, including the presence of enamel spindles. Scanning electron microscopy was performed on mid-tooth sections of five maxillary CT, five mandibular CT and two incisors. The ultrastructural anatomy of primary and secondary dentine, and equine enamel types-1, -2 and -3 (as described in horses) were identified in donkey teeth. Histological and ultrastructural donkey dental anatomy was found to be very similar to equine dental anatomy with only a few quantitative differences observed.

Post-mortem examination of 19 donkey skulls showed that donkeys have a greater degree of anisognathia (27% width difference between upper and lower jaws) compared to horses (23%). Teeth (n=108) were collected from 14 skulls and examined grossly and by computed axial tomography (CAT). A greater degree of peripheral enamel infolding was found in mandibular cheek teeth (CT) compared to maxillary CT (P<0.001). A significant increase in peripheral cementum from the apical region to the clinical crown was demonstrated in all CT (P<0.0001). All donkey CT had at least five pulp cavities with six pulp cavities present in the 06s and 11s. A new endodontic numbering system for equid CT has been proposed. A greater occlusal depth of secondary dentine (mm) was present in older donkeys (>16 years) than in the younger (<15 years) donkeys studied. Based on gross and CAT examinations, donkey dental anatomy was shown to be largely similar to that described in horses.

Background: Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellular hypertonic stress. Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine transport., Methods: We studied the possible role of microtubules in this adaptive mechanism using renal cells in culture. RESULTS.: In cultured renal cell lines [Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)], up-regulation of BGT1 activity was maximal after 24 to 30 hours in growth medium made hypertonic (510 mOsm/kg) by the addition of sucrose or NaCl. Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium. Both cycloheximide (20 micromol/L) and nocodazole (20 micromol/L) blocked the hypertonic up-regulation of BGT1. Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium. Recovery from nocodazole action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking to the cell surface once microtubules repolymerized. Hypertonic activation of BGT1 transport was detected in an isolated membrane fraction and was blocked by cycloheximide but not by nocodazole. Confocal microscopy confirmed the increased abundance of BGT1 proteins in the plasma membrane of hypertonic cells and showed that BGT1 remained intracellular during nocodazole treatment., Conclusions: Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional transporters to the cell surface. The apparent resistance of membrane BGT1 to nocodazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters.

Two groups of eight Holstein-Friesian heifers were fed either a grass-silage-based diet (S) or one based on meadow hay supplemented with 1.8 kg/day barley concentrate mix (H) during cubicle housing as young stock (and in early pregnancy). Lameness and claw lesion development were monitored from approximately four weeks before until 20 weeks after first calving. No significant difference was found between S and H for claw conformation or horn growth and wear. Both groups showed net wear immediately after calving. The prevalence of poor locomotion and the extent of lesion development 20 weeks after calving (when they were highest) were significantly (P< 0.05) higher in S than H. It was concluded that feeding grass silage to young stock may deleteriously affect subsequent claw health and that this risk factor requires further study., (Copyright 2000 Baillière Tindall.)

A series of polyester fiber ACL implants was studied in ovine stifle joints up to 2 years postimplantation. The implants were linked to the bone-tunnel wall by oriented fibrous tissue. Cross-sections of the tunnels showed bone ingrowth among the implant fibers at 2 years. A human trial of the Apex implant yielded a series of retrievals, some associated with gross bone-tunnel enlargement. There was no evidence of bone ingrowth in the human implants. It was hypothesized that-tunnel enlargement resulted from fretting at the implant-tissue interface in response to cyclic loads in use.

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 microM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ATPase activity in whole cells, Na+K+ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

The permeability barrier in the dorsal wall of the equine hoof capsule was studied by means of horseradish peroxidase (HRP) in 0.9 N saline solution as a water soluble tracer. Section were treated with 3'3'-diaminobenzidine tetrachloride (DAB) and before dissection the quality of the horn of feet from 10 horses was assessed and given a subjective grade as either good or poor. Blocks of tissue from each horse were left in either an oven at 60 degrees C or in water for 2 weeks before treatment in HRP, sectioning and DAB solution. Regions observed were i) outer surface, ii) outermost layers of the horn, iii) cut edge of the outer layer, iv) inner layer of horn, v) cut edge of the inner layer and vi) laminae. Horn deemed to be normal horn and of good 'quality' showed very slight penetration of HRP 3-5 cell layers deep in the outer layer. The cut edge of the outer layer of the wall of the 'normal' horn also showed minimal penetration of HRP through the intercellular spaces. The cut edge of the inner layers of the wall of normal, good quality horn showed penetration of the tracer up to 20 cell layers deep, with HRP in both the intercellular spaces and within the cells. In contrast, sections of horn from horses with brittle feet showed deep cracks in the outer surface into which the HRP had penetrated. Good quality horn showed no change in the position of the permeability barrier after soaking in water for 14 days, but the brittle horn showed an increase in permeability to HRP. In brittle horn, reaction product was seen deep within the section in the intercellular spaces of the intertubular horn only. Placing horn in an oven had no effect on the permeability barrier. The permeability barrier of the dorsal wall of the equine hoof capsule differs with the layer of the wall. Horn considered to be of poor quality had a weaker permeability barrier than horn of good quality.

Accumulation of osmolytes by renal cells is due in part to increased uptake via specific transporters. These include amino acid transport system A and the betaine/GABA transporter (BGT1). Transport changes have been characterized using intact cells which makes the intracellular mechanisms difficult to determine. In this study the hypertonic upregulation of system A and BGT1 was studied directly at the membrane level in Madin-Darby canine kidney (MDCK) cells. Both system A and BGT1 transport systems were detected in an isolated membrane fraction containing plasma membranes. System A transport was increased in membranes prepared from cells after 6 h hypertonic stress (449 mosmol/kg) but BGT1 activity was minimal and not different from isotonic controls. The increase in system A was blocked by inhibitors of RNA and protein synthesis. BGT1 transport was induced in membranes prepared after 24 h hypertonicity. At this time system A activity in the membrane fraction remained increased, unlike the downregulation observed in intact MDCK cells. We conclude that differential upregulation of system A and BGT1 by hypertonic stress is due to intrinsic changes in these transporters at the membrane level. In contrast, the downregulation of system A in intact cells when hypertonicity is prolonged for 24 h is likely due to the action of an intracellular repressor that is not present in the isolated membranes., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Claw lesions are a major cause of lameness in dairy cattle. Analysis of the development of lesions is aided by numerical representation of their significance. Using data from observations on 31 heifers at 9 weeks post-calving, 5 lesion scoring method were compared. These were: (1) number of lesions; (2) severity; (3) adjusted severity; (4) size (measured by a novel technique involving image analysis of distal view photographs) and (5) size multiplied by adjusted severity (combined score). Relationships between scores for sole and white line lesions and between different claws within a cow were investigated. The small size but high clinical significance of severe lesions means that severity must be weighted if combined with size in a score. Sole and white line lesions showed a moderate but significant correlation in terms of severity but none in terms of size. The highest correlation between scores for a single claw (the right hind outer) and the remaining claws was found for adjusted severity of sole lesions.

The development of claw haemorrhages was monitored in first-calving dairy heifers from 4 weeks before calving to 32 weeks after calving. Before calving, lesions were few but the number, extent and severity of claw haemorrhages increased following simultaneous calving and housing. Lesions were most severe in the white line at 9 weeks, and in the sole at 14 weeks, but recovery began while the animals were still housed. That the increase in white line lesions after calving, and the subsequent recovery preceded that of the sole, suggests that the pathogenesis of the lesions may differ in these two anatomical regions. It is proposed that an initial insult to the corium primarily affects the laminar region and that corium damage increases with the resulting alteration in the physical forces on the sole.

Cultured vascular endothelial cells take up L-proline by sodium-dependent transport. Cells incubated in medium made hyperosmotic by addition of sucrose showed a dose-dependent increase in Na+/proline cotransport. Studies with alpha-(methylamino)isobutyric acid revealed that the up-regulation was specific for amino acid transport system A. Up-regulation was blocked by actinomycin D and cycloheximide, indicating roles for gene transcription and protein synthesis. Up-regulation was maximum after five to six hours of hyperosmotic treatment, but returned to control levels when osmotic stress was maintained for 24 hours. The decline at 24 hours was accompanied by a significant increase in Na+/gamma-aminobutyric acid cotransport. The activity of this system, which also transports betaine, remained unchanged after just five hours of hyperosmotic stress. Inclusion of betaine in the hyperosmotic medium reduced up-regulation of system A. Na/Pi cotransport also was up-regulated by five hours of hyperosmotic stress. Up-regulation of system A, but not Na/Pi cotransport, was detected in isolated membrane fractions indicating that increased activity of this membrane transport system may be one mechanism by which vascular endothelial cells accumulate amino acids. The amino acids may act as organic osmolytes to help maintain normal cell volume during the early phase of hyperosmotic stress.

Chronic renal adaptation to dietary deprivation of Pi is accompanied by increased Na+/Pi co-transport across the brush border membrane of the renal proximal tubule. The increased activity of this co-transport system depends on de novo protein synthesis and insulin. The present study used normal and diabetic rats to determine if the endosomal pool of Na+/Pi co-transporters was altered by Pi deprivation and the possible role of insulin. In response to 5 days of dietary Pi deprivation there was a significant increase in endosomal Na+/Pi co-transport in control rats but there was no change in diabetic rats. The increase in endosomal Pi uptake was restored in diabetic rats treated with exogenous insulin. Na(+)-independent Pi uptake and proline uptake remained unchanged in all groups. The changes in endosomal Na+/Pi co-transport correlated with the abundance of the specific Na+/Pi co-transporter protein, as determined by Western blots. The pattern of endosomal changes paralleled that observed in brush border membranes. One possibility consistent with these findings is that the endosomal fraction contains newly synthesized Na+/Pi co-transporters targeted for delivery to the apical brush border membrane. Increased synthesis and delivery is required to maintain the adaptation to chronic Pi deprivation.

Inorganic phosphate (Pi) is reabsorbed in renal proximal tubules in a sodium (Na)-dependent manner involving brush border Na/Pi-cotransporter(s). Regulation of renal Pi-reabsorption, such as by parathyroid hormone (PTH) and/or by dietary Pi-deprivation, involves alterations in the rate of Na/Pi-cotransport. Two structurally different Na/Pi-cotransporters have been identified: type I-transporter and type II-transporter. The related mRNAs and proteins are located in the proximal tubule and in the brush border membrane. In heterologous expression systems type I and type II Na/Pi-cotransporters mediate Na/Pi-cotransport. Characterization of the transport properties suggested that the type II transporter is "responsible' for brush border membrane Na/Pi-cotransport (as observed in isolated vesicles). Administration of PTH to rats resulted in an inhibition of brush border membrane Na/Pi-cotransport (vesicles) and in a reduced brush border membrane content of the type II transporter. Feeding low Pi-diets resulted in an up-regulation of Na/Pi-cotransport (vesicles) and of type II transporter content; only after a prolonged exposure to low Pi-diets (more than 4 hr) was an increase in specific mRNA content observed. Refeeding high Pi diets had the opposite effects on Na/Pi-cotransport activity and on type II transporter protein. It is currently the task of future experiments to define the specific mechanisms leading to protein-synthesis-independent (PTH, acute Pi-deprivation, Pi-refeeding) and to protein-synthesis-dependent (prolonged Pi-deprivation) regulation of the type II Na/Pi-cotransporter.

Neutral amino acids are important organic osmolytes found in most osmotically stressed cells. During prolonged hypertonic stress, accumulation of neutral amino acids contributes to the cell volume recovery and may protect cellular proteins against salt denaturation. Hypertonicity activates system A transport, a major neutral amino acid transporter in mammalian cells, by a microtubule-dependent mechanism. Similar to the regulation of amino acid efflux induced by hypotonic-swelling, influx via system A may be under the control of intracellular ionic strength. An osmotically sensitive repressor may negatively regulate the gene expression of a regulatory protein which activates the preexisting system A transporter protein. Hypertonically activated protein kinases may be involved in this osmoregulation of amino acid transport.

Parathyroid hormone (PTH) inhibits Na(+)-dependent Pi uptake in renal epithelial cells from opossum kidney (OK). This requires an intact endocytic pathway, suggesting that one action of PTH may be to promote endocytic removal of Na+/Pi cotransporters from the cell membrane. The present study tested if PTH, at a dose that inhibited membrane Pi transport, also produced an increase in endocytic activity. Pi transport was measured in isolated plasma membrane vesicles. Endocytosis was measured by allowing cells to take up horseradish peroxidase (HRP) followed by assay of triton-sensitive (latent) HRP activity in subcellular fractions isolated by density gradient centrifugation. Incubation of OK cells with 10(-7) M PTH for 3 h decreased Na+/Pi cotransport by membrane vesicles to 328 +/- 54 pmol/mg/min compared to 448 +/- 67 pmol/mg/min (mean +/- S.E., P < 0.03) in controls. Latent HRP content of endosomal fractions was dependent on the time and temperature used to load cells with HRP and on the concentration of HRP. However, incubation of OK cells with 10(-7) M PTH for either 1 or 3 h produced no change in latent HRP activity. Thus the action of PTH on the Na+/Pi cotransporter in the plasma membrane of OK cells does not require a change in the rate of endocytosis.

Insulin stimulates reabsorption of phosphate (Pi) in the renal proximal tubule. Previous studies have shown that vanadate can mimic the action of insulin on various tissues. In the present study, we tested the action of vanadate on renal Pi transport both in control rats and in rats made diabetic by injection of streptozotocin. Vanadate was administered orally for 4 days by inclusion in drinking water (0.7 mg/ml). By the 4th day, vanadate treatment of control rats did not change acid-base status, plasma glucose or the filtered load of Pi, but the urinary excretion of Pi was reduced to 2.5 +/- 0.9 compared with 17.6 +/- 3.5 mumol/mg creatine (P < 0.02) in untreated control rats. However, Na+/Pi cotransport by isolated brush border membrane vesicles was not different between the two groups. Findings in parathyroidectomized rats were similar. By the 4th day of vanadate treatment of diabetic rats, there was reversal of polyuria, polydipsia and hyperglycemia with no change in acid-base status. The filtered load of Pi was decreased by vanadate, and urinary Pi excretion also tended to decrease but not significantly. The values for Pi excretion were 21.4 +/- 7.6 in vanadate treated diabetics and 36.1 +/- 4.5 mumol/mg creatinine in untreated diabetics. In contrast to vanadate, daily injections of insulin did not change the filtered load of Pi but reduced urinary Pi excretion in diabetic rats to 15.6 +/- 2.2 mumol/mg creatinine (P < 0.02). These findings suggest that vanadate stimulated tubular Pi reabsorption in control rats but not in diabetic rats. Vanadate treatment of diabetic rats may tend to decrease tubular Pi reabsorption in contrast to the action of insulin.

Renal proximal Na/Pi-cotransport is increased in response to low dietary Pi intake. Recently, a cDNA (NaPi-2) related to the rat renal brush membrane Na/Pi-cotransporter has been cloned. In the present study, we used rats fed for 6 days with either a low Pi diet (LPD) or a high Pi diet (HPD), respectively. In parallel to an increased renal brush-border membrane Na/Pi-cotransport in LPD rats, there was also an increased content of NaPi-2 mRNA in renal cortex. After injection into Xenopus laevis oocytes, mRNA isolated from LPD rats induced a greater increase in Na/Pi-cotransport compared to mRNA from HPD rats. Hybrid depletion experiments suggested that mRNA-induced Na/Pi-cotransport is related to NaPi-2. We conclude that chronic Pi deprivation leads to an increased brush-border membrane Na/Pi-cotransport via an increase in the level of (NaPi-2) mRNA.

Deprivation of dietary phosphate leads to a decrease in urinary excretion of ammonium in rats which may be mediated through an alteration in ammonium transport in the proximal tubule. In the present study the OK renal epithelial cell line, a model for the proximal tubule, was used to determine if NH4+ transport was changed during acute phosphate deprivation. The intracellular pH after perfusion with NH4Cl solution was used for calculation of intracellular NH4+ concentration. Intracellular [NH4+] increased linearly during the first 2 min of acidification with NH4Cl. NH4+ transport, defined by the initial rate of the increase in intracellular [NH4+], was decreased by 32% (P < 0.005) in phosphate-deprived cells. This transport process was inhibited by barium chloride, but not by DIDS, amiloride or ouabain, suggesting that the NH4+ transport pathway may utilize K+ channels. Acute phosphate deprivation may inhibit NH4+ transport in OK cells by decreasing membrane K+ permeability.

The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolayer culture these cells transport inorganic phosphate and L-alanine via Na(+)-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60-70 per cent increase in Na(+)-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na(+)-dependent alanine uptake and Na(+)-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased Vmax and was blocked completely by pretreatment with cycloheximide (70 microM). In these cells recovery of intracellular pH after acidification with NH4Cl is due primarily to the Na+/H+ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na+/H+ exchange was inhibited during phosphate deprivation.

An endosome-enriched fraction was prepared from rat kidney cortex by a standard procedure employing centrifugation on a Percoll gradient. This fraction showed time-dependent accumulation of inorganic phosphate (Pi) which was stimulated two- to threefold during the initial phase by an inwardly directed Na+ gradient. Na+ gradient-dependent Pi accumulation decreased with increasing medium osmolality and Pi binding accounted for only 16% of the total accumulation at two minutes. Like the Pi transporter in the brush border membrane (BBM), the Na+ gradient-dependent Pi uptake (but not the Na(+)-independent component) by the endosomal fraction was stimulated by intravesicular Pi and by an outwardly directed proton gradient, and was inhibited by extravesicular arsenate. Unlike the Pi transporter in BBM, the endosomal Pi transporter was not changed by acidic pH under non-gradient conditions. Activation of the endosomal proton pump by extravesicular ATP, leading to acidification of the vesicle interior, was accompanied by stimulation of endosomal Na+ gradient-dependent Pi transport. Inhibition of the proton pump by deletion of chloride or addition of N-ethylmaleimide abolished the stimulation of Pi uptake by ATP. The data indicate that the Na(+)-dependent Pi transporter in renal endosomal fractions is an intrinsic endosomal component. It remains to be determined if the endosomal Pi transporter plays a role in regulation of renal Pi transport.

We excised the anterior cruciate ligament from the left stifle of 24 sheep and replaced it by a polyester fibre implant routed 'over the top' of the femoral condyle and fixed, using grommets and screws. All the joints were sound, and the animals moved normally until they were killed at six, 12 and 24 months after operation. We found that the implants were always covered by host tissue, which matured into bundles with a histological appearance similar to the natural ligament. The implants were joined to the bones by organised fibrous tissue and there was no anchorage loosening. There was no synovitis, but the operated joints showed progressive cartilage degeneration. The reconstructed joints became less stable immediately after operation, but regained normal stability as the neoligaments developed. The neoligaments lost strength with time, despite tissue ingrowth. The good functional, biomechanical, and histological results justify clinical trials of this type of implant.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.

When grown in monolayer culture, OK cells display endocytic uptake of soluble fluid-phase markers such as lucifer yellow (LY) and horseradish peroxidase (HRP). The response of this process to metabolic inhibitors was characterized in the present study. Inhibition of cell metabolism by cyanide produced a decrease in cell ATP content which was accompanied by a decrease in uptake of both LY and HRP, confirming the energy-dependence of fluid-phase endocytosis in OK cells. Use of iodoacetate also decreased cell ATP content but its action on endocytosis was unexpected. Cell uptake of HRP was decreased by iodoacetate, similar to the effect of cyanide, but there was a marked increase in LY uptake. Additional studies showed that cyanide did not change intracellular Na+ or intracellular K+ and did not interfere with the Na(+)-dependency of Pi uptake. In contrast, iodoacetate produced a marked increase in Na+, a decrease in K+, and abolished the Na(+)-dependency of Pi transport. The latter was due primarily to a 10-fold increase in Na(+)-independent uptake of Pi. These findings suggest, indirectly, that plasma membrane permeability to Na+, K+, Pi, and small molecules such as LY, may be increased by iodoacetate, possibly through its action as an alkylating agent. This mechanism may allow increased cell uptake of LY through a non-endocytic pathway, and may mask the inhibitory action of iodoacetate on endocytic uptake of LY. These additional effects complicate the use of iodoacetate to interrupt endocytosis.

Farrier's Formula feed supplement was added to the diet of 18 horses with two types of hoof horn defects. The first group of horses showed sand cracks and crumbling horn around the nail holes; the second group suffered frequent bruising and had flat feet with collapsed heels. Hoof clippings from both groups were studied in the transmission and scanning electron microscopes. All the horses showed a progressive improvement in the gross and microscopic structure of the hoof horn, starting six weeks after the supplementation began. Once good quality hoof horn had grown there was no relapse during the two year period of the study.

Suspensions of proximal tubules were obtained by collagenase digestion of rat renal cortex followed by centrifugation on a percoll gradient. NAD content in tubules incubated at 37 degrees C was decreased by 40-60% compared with tubules incubated at 4 degrees C. This change occurred within 30 min and was maintained for up to 2 hr. Inhibitors of NAD hydrolysing enzymes prevented the depletion of cellular NAD at 37 degrees C. Acute changes in proximal tubule NAD content at 37 degrees C were not accompanied by changes in phosphate uptake by brush border membrane vesicles subsequently prepared from the same tubules. In contrast, incubation of tubules with parathyroid hormone (10(-6) M) produced the expected inhibition (20%) of brush border membrane transport of phosphate. One implication of these findings is that acute changes in total NAD content of proximal tubules at 37 degrees C may not influence the phosphate transport system in the renal brush border membrane. Other interpretations are discussed.

Endocytosis was studied in OK epithelial cells, an established cell line from opossum kidney. The presence of fluid-phase endocytosis in these cells was demonstrated by measuring cell uptake of lucifer yellow and horseradish peroxidase. The intracellular distribution of lucifer yellow fluorescence was consistent with uptake by endocytosis. Endocytosis was inhibited in medium made hyperosmolar by addition of sucrose. In hyperosmolar medium the action of parathyroid hormone on Na+/phosphate cotransport was significantly diminished. We suggest that an intact endocytic mechanism is required for the full inhibitory effect of parathyroid hormone on Na+/phosphate cotransport.

Autosomal dominant polycystic kidney disease is characterized by the formation of large fluid-filled epithelial cysts. To obtain renal cyst wall epithelium for in vitro study, we employed an explant culture technique using medium-hydrated collagen gel as the culture substrate. Pieces of excised cyst wall were submerged within collagen gel. Cells of the cyst lining migrated to form a polarized epithelium at the surface of the surrounding collagen gel. Regions of the outgrowth were isolated by microdissection and used as a source of cells for subculture. Cyst-derived epithelium retained the ultrastructural features of the renal cyst of origin. The cells were cuboidal and bore short apical microvilli. Cells often were joined by apical tight junctions. Intercellular channels were narrow and bordered by short microvillus projections at the basolateral membrane. Epithelial cells rested on a densely staining basal lamina. The explants commonly developed small solitary cysts within their wall that were filled with fluid. These mural cysts were lined by a simple epithelium morphologically similar to the cells that lined the explant. Explantation of human renal cyst wall to culture within collagen gel provides a reproducible method to isolate autosomal dominant polycystic kidney disease epithelium for subculture. This method offers an alternative to the use of proteolytic enzymes to establish morphologically stable cultures of cyst lining cells for use in experimental renal cystic disease.

Administration of nicotinamide to rats produces specific dose-dependent inhibition of Na+-dependent phosphate transport across the renal brush-border membrane (BBM) and an increase in urinary excretion of phosphate. The intracellular mechanism of action of nicotinamide is not well established. As a step in this direction, the present studies determined whether nicotinamide was a rapid- or slow-acting regulator of the BBM phosphate transport system. Nicotinamide (0.5 g/kg) inhibited Na+-dependent BBM phosphate transport under conditions when de novo protein synthesis was inhibited by cycloheximide (1.0 mg/kg). Furthermore, the degree of inhibition was not different from that achieved by nicotinamide alone, suggesting that the action of nicotinamide does not require de novo protein synthesis. Studies on the time course of the onset of nicotinamide action revealed inhibition of BBM phosphate transport within 1 h after injection of nicotinamide, even in rats pretreated with cycloheximide. The rapid response to nicotinamide and its independence of de novo protein synthesis characterize nicotinamide as a rapid-acting regulator of the Na+-dependent phosphate transport system in renal BBM.