Static compressive loading reduces the mRNA expression of type II and X collagen in rat growth-plate chondrocytes during postnatal growth.

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.

The effects of mechanical loading on the mRNA expression of growth-plate cells.

Bone growth is a complex process involving proliferation, maturation and hypertrophy of chondrocytes in the growth plates. Mechanical forces applied to growing bones alter their longitudinal growth. However, the mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of mechanical loading on the mRNA expression pattern of key molecular components of the growth-plate. Short-term static loading was applied to rat proximal tibial growth-plate explants. Various age groups at specific developmental stages were investigated. In situ hybridization was used to assess the mRNA expression of the cells in different zones of the growth-plate. Four key components were investigated: 18s (basic cell metabolism), type II collagen (major extracellular matrix component), type X collagen (matrix component in hypertrophic zone) and PTH-PTHrP receptors (pre-hypertrophic chondrocytes). The spatial variation in the mRNA expression between loaded explants and their contralateral controls was compared to establish: -the sensitivity of the different growth-plate zones to mechanical loading; -the sensitivity of the different developmental stages to loading. Preliminary results indicated that static loading on the growth plate of 80 d.o. rats affects type II and X collagen gene expressions while PTH-PTHrP remains insensitive to static loading. Improved understanding of growth-plate mechanics and the underlying biology is required to provide a scientific basis for the treatment of progressive deformities.

Macromolecular transport across the rabbit proximal and distal colon.

BACKGROUND: Although many studies have investigated macromolecular uptake in the stomach and small intestine, little is known about macromolecular uptake in the colon. AIMS: To investigate the mechanisms involved in the transport of large antigenically intact macromolecules across the proximal and distal colonic epithelium in the rabbit. METHODS: The mucosal to serosal movement of bovine serum albumin (BSA) was examined in modified Ussing chambers under short circuited conditions. The mucosal surface was exposed to varying concentrations of BSA, and after a 50 minute equilibration period, the mucosal to serosal flux of immunologically intact BSA was determined by ELISA. Total BSA flux was determined by the transport of radiolabelled 125I-BSA. RESULTS: Intact BSA transport in proximal and distal colonic tissue showed saturable kinetics. Intact BSA transport in the proximal and distal segment was 7% and 2% of the total 125I-BSA flux respectively. Immunologically intact BSA transport in the distal segment was significantly less than that in the proximal segment. Intact BSA transport in the proximal colon was significantly reduced following treatment with sodium fluoride, colchicine, and tetrodotoxin. Cholinergic blockade had no effect on the uptake of intact BSA. CONCLUSION: The findings indicate that the transport of intact macromolecules across the proximal and distal large intestine is a saturable process. Further, intact BSA transport in the proximal colon is an energy dependent process that utilises microtubules and is regulated by the enteric nervous system.

Mast cell hyperplasia and increased macromolecular uptake in an animal model of giardiasis.

Giardiasis has been associated with an increase in allergic disease following infection suggesting an alteration in mucosal immune function. Jejunal in vivo and in vitro macromolecular transport, epithelial permeability, and mucosal and connective tissue mast cell counts were examined in Mongolian gerbils (35-45 g) orogastrically inoculated (I) with a pathogenic strain of Giardia lamblia and compared to age- and weight-matched, sham-treated controls (C) 6 and 21 days postinoculation. Macromolecular uptake was significantly increased in infected tissue at 6 days both in vivo (I 134 +/- 19 vs. C 74 +/- 17 ng/hr; n = 8; P < 0.05) and in vitro (I 125 +/- 17 vs. C 67 +/- 8 ng/hr/cm; n = 12; P < 0.05). Macromolecular uptake did not differ between groups at 21 days. Infection had no effect on mucosal permeability of [51Cr]EDTA. Mucosal mast cell counts did not differ at 6 days but were significantly elevated in infected tissue at 21 days (I 33.3 +/- 6.8 vs. C 2.7 +/- 0.4 per high magnification field; n = 5; P < 0.01) as were connective tissue mast cell counts (I 1.7 +/- 0.2 vs. C 1.0 +/- 0.1 per high magnification field; n = 13; P < 0.005). The findings indicate that during the peak phase of giardiasis, jejunal active antigen uptake is increased leading to a delayed recruitment of mucosal and connective tissue mast cells. These changes may play a role in the increased incidence of hypersensitivity reactions associated with Giardia infection.

Transport of albumin into the intestinal lumen of the rat.

The intestine is considered a major site for the breakdown and clearance of serum proteins. The mechanism of transport of macromolecules from the serosa into the lumen is unclear. The present study was designed to characterize the serosal to mucosal movement of albumin. Transport of bovine serum albumin (BSA) was assessed in short-circuited Ussing chambers, using stripped rat jejunum devoid of Peyer's patches. To define the kinetics of serosal to mucosal albumin transport the serosal surface was exposed to BSA at varying concentrations (0.5-5 mg.mL-1). Fluids from the mucosal compartment were sampled over time and assayed for immunologically intact BSA by ELISA. All subsequent experiments utilized a concentration of cold BSA (2 mg.mL-1) that produced maximal levels of intact BSA transport. To assess total BSA transport (intact BSA plus degraded BSA), 10 microCi (1 Ci = 37 GBq) 125I-labelled BSA was added to the serosal surface in addition to 2 mg.mL-1 cold BSA. To further characterize BSA transport tissues were treated with sodium fluoride (NaF) (metabolic inhibitor) or colchicine (an inhibitor of microtubule polymerization) or with the nerve blocker tetrodotoxin (TTX). All experiments using inhibitors were performed in paired tissues obtained from the same animal. Transport of intact BSA into the intestinal lumen was a saturable process, with a Vmax of 251 +/- 13 ng.cm-2.h-1 and a Km of 0.72 +/- 0.1 mg.mL-1, and represented 7% of the total BSA flux into the intestinal lumen. In the presence of NaF (2 x 10(-3) M), transport of both intact and total BSA was significantly inhibited (intact: control 374 +/- 80 vs. NaF 46 +/- 11, 88% inhibition; total: control 3288 +/- 296 vs. NaF 2550 +/- 235 ng.cm-2.h-1, 22% inhibition; p < 0.05). In addition, colchicine significantly inhibited intact BSA transport (control 339 +/- 15 vs. colchicine 206 +/- 13 ng.cm-2.h-1, 39% inhibition; p < 0.05). TTX had no effect on intact BSA flux. The findings suggest that transport of intact BSA from the serosa into the intestinal lumen is a saturable, energy-dependent process, which involves microtubules but is not under neural regulation.

The role of nitric oxide in the regulation of macromolecular transport in rat jejunum.

1. Nitric oxide is known to affect epithelial and microvascular permeability and is a major non-adrenergic non-cholinergic neurotransmitter in the intestine. We have previously demonstrated neuronal regulation of macromolecular transport in the intestine. To define this regulation further the role of nitric oxide was investigated. 2. Stripped rat jejunum was mounted in Ussing chambers exposing the mucosal surface to bovine serum albumin (BSA; 2 mg ml-1), or BSA (2 mg ml-1) plus [125I]BSA (10 microCi). Following a 50 min equilibration, serosal fluids were sampled for four 10 min periods, and fluxes determined for intact BSA by enzyme-linked immunosorbent assay (ELISA) and total BSA by [125I]BSA under basal conditions, and after treatment with NG-nitro-L-arginine-methyl ester (L-NAME) alone or in conjunction with L-arginine or decarboxylated molsidomine (SIN 1). 3. L-NAME significantly increased intact BSA uptake. Total (intact + degraded) BSA flux was not altered. The L-NAME effect was reversed by L-arginine and SIN 1. Additional experiments were performed by adding the nitric oxide donors sodium nitroprusside and SIN 1 directly to control tissue. Nitric oxide donors did not further decrease intact BSA flux below levels obtained from control tissue. The L-NAME enantiomer D-NAME had no effect. Sodium-free bathing solutions also had no effect on intact BSA uptake. Non-specific permeability, as assessed by the serosal to mucosal movement of [51Cr]ethylene-diamine-tetraacetate ([51Cr]EDTA), was decreased with L-NAME. 4. The findings indicate that nitric oxide downregulates intact macromolecular flux in the small intestine.

Transport of bovine serum albumin across rat jejunum: role of the enteric nervous system.

To assess the mechanisms for movement of antigenically intact macromolecules across small intestinal mucosa, transport kinetics of bovine serum albumin (BSA) uptake and the effect of neural and metabolic inhibition were examined in stripped short-circuited rat jejunum. The mucosa was exposed to BSA, and, after a 50-min equilibration, mucosal-to-serosal movement of immunologically intact BSA was determined by enzyme-linked immunosorbent assay and total BSA by radiolabeled 125I-BSA. Intact BSA uptake demonstrated saturable kinetics. Immunologically intact BSA crossed the intestinal mucosa as 4.5% of total 125I-BSA flux. Colchicine and 4 degrees C significantly reduced uptake of immunologically intact BSA. NaF significantly reduced uptake of immunologically intact BSA and 125I-BSA. Treatment with tetrodotoxin significantly reduced intact BSA uptake, but did not significantly alter total BSA uptake. The muscarinic cholinoceptor antagonist atropine also significantly inhibited transport of intact BSA, whereas the nicotinic cholinoceptor antagonist hexamethonium had no effect. These findings indicate that transport of intact macromolecules across small intestinal mucosa is a saturable energy-dependent process that utilizes the microtubular network and is regulated by the enteric nervous system primarily through cholinergic nerves acting on muscarinic receptors.