Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth μscle cells in culture

Opsahl, William; Ehrhart, L A

doi:10.1042/bj2430137

Cited by 26 publications

(13 citation statements)

References 34 publications

(50 reference statements)

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“…When two AA tracers are used for skin protein measurement, as in the present experiment, an additional issue is that, if they have a different relationship between tissue fluid and tRNA enrichment, the use of tissue fluid as precursor enrichment could result in inconsistent FSR values. It is likely that possible compartmentalization of Pro pools (18) explains why the FSR values in epidermal and dermal protein measured from Phe and Pro tracers were different (see Table 3), although both showed that epidermal FSR was much greater than dermal FSR. In the present experiment, we also measured skin protein FSR from a leucine tracer.…”

Section: Discussionmentioning

confidence: 99%

Measurement of protein metabolism in epidermis and dermis

Zhang

Chinkes

Wolfe

2003

American Journal of Physiology-Endocrinology and Metabolism

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; 10.1152/ajpendo. 00460.2002.-We found that, in the rabbit ear, the dermal protein contains 75.5% of cutaneous phenylalanine and 97.9% of cutaneous proline; the remaining 24.5% of phenylalanine and 2.1% of proline are in the epidermal protein. This finding led us to develop two novel models that use phenylalanine and proline tracers and the rabbit ear to quantify protein kinetics in the epidermis and dermis. The four-pool model calculates the absolute rates of protein kinetics and amino acid transport, and the two-pool model calculates the apparent rates of protein kinetics that are reflected in the blood. The results showed that both epidermis and dermis maintained their protein mass in the postabsorptive state. The rate of epidermal protein synthesis was 93.4 Ϯ 37.6 mg ⅐ 100 g Ϫ1 ⅐ h Ϫ1 , which was 10-fold greater than that of the dermal protein (9.3 Ϯ 5.8 mg ⅐ 100 g Ϫ1 ⅐ h Ϫ1 ). These synthetic rates were in agreement with those measured simultaneously by the tracer incorporation method. Comparison of the four-pool and two-pool models indicated that intracellular cycling of amino acids accounted for 75 and 90% of protein kinetics in the dermis and epidermis, respectively. We conclude that, in the skin, efficient reutilization of amino acids from proteolysis for synthesis enables the maintenance of protein mass in the postabsorptive state. stable isotopes; gas chromatograph and mass spectrometer; arteriovenous balance; fractional synthesis rate; rabbits THE SKIN IS ONE OF THE LARGEST TISSUES in the body. In normal circumstances, the skin maintains a constant protein mass, whereas skeletal muscle may have a significantly positive or negative protein balance (28). The homeostasis of skin protein mass plays a crucial role in preserving an intact barrier on the body surface. Once the integrity of the skin barrier is destroyed, several problems ensue immediately, such as loss of water and electrolytes, invasion of microorganisms, and impairment of immune functions (11,19,20). Thus knowledge of skin protein metabolism is important in understanding the maintenance and repair process of the skin barrier.In our previous experiments (28-30), we investigated protein metabolism in normal and scalded skin by use of a rabbit ear model. This model has been the only approach to simultaneously quantify the in vivo rates of protein synthesis and breakdown as well as amino acid (AA) transport in the skin. However, this model is limited by the consideration of the skin as a single pool. It is well known that the epidermis and dermis have different structure, function, and turnover rates (12,17,25). The dermis serves as a basis for supporting and nourishing the epidermis, whereas the epidermis exerts the barrier function of the skin. It is therefore important to distinguish between the metabolic regulation of the epidermis and that of the dermis under physiological and pathological conditions.The goal of this study was to establish an approach for quantitation of protein kinetics and AA transport in the epidermis and dermis. This...

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Section: Discussionmentioning

confidence: 99%

Measurement of protein metabolism in epidermis and dermis

Zhang

Chinkes

Wolfe

2003

American Journal of Physiology-Endocrinology and Metabolism

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“…This may be partially explained by the slow turnover of collagen, which is only 1.3%/day in rat skeletal muscle under control conditions [36]. It has also been shown that a great proportion of newly synthesized soluble collagen is degraded without being deposited [33]. In human muscle injury induced by strenuous exercise for 7 days, muscle hydroxyproline concentration is elevated until 2 months post-exercise [12].…”

Section: Discussionmentioning

confidence: 99%

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running

Han¹,

Wang²,

Komulainen³

et al. 1999

Pfl�gers Archiv European Journal of Physiology

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The purpose of the study was to investigate pre-translational regulation of collagen expression after a single bout of exercise. We analysed steady-state messenger ribonucleic acid (mRNA) levels for collagen types I, III and IV, alpha- and beta-subunits of prolyl 4-hydroxylase and lysyl oxidase (enzymes modifying procollagen chains), and enzyme activity of prolyl 4-hydroxylase from rat soleus muscle (MS) and the red parts of quadriceps femoris muscle (MQF) after 12 h and after 1, 2, 4, 7 and 14 days of downhill (-13.5 degrees ) treadmill running at a speed of 17 m.min-1 for 130 min. Histological and biochemical assays revealed exercise-induced muscle damage in MQF but not MS. Steady-state mRNA levels for the alpha- and beta-subunits of prolyl 4-hydroxylase in MQF, lysyl oxidase in MS and MQF were increased 12 h after running, whereas prolyl 4-hydroxylase activity did not increase until 2 days after exercise. The mRNA levels for the fibrillar collagens (I and III) and basement membrane type IV collagen significantly increased 1 day and 12 h after exertion, respectively. Peak mRNA levels were observed 2-4 days after running, the increases being more pronounced in MQF than in MS. No significant changes were observed in types I or III collagen at the protein level. Strenuous downhill running thus causes an increase in gene expression for collagen types I and III and their post-translational modifying enzymes in skeletal muscle in a co-ordinated manner. These changes, together with the increased gene expression of type IV collagen, may represent the regenerative response of muscle extracellular matrix to exercise-induced injury and an adaptive response to running exertion.

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“…21 ' 22 Cells were grown in Dulbecco's modified Eagle's/Ham's F12 (DME/F12) medium (Irvine Scientific, Irvine, Calif.) that contained 0.12% NaHCOj and 10% fetal bovine serum (FBS). Amino acid supplementation resulted in a final concentration of 0.25 mM L-proline.…”

Section: Cell Culturementioning

confidence: 99%

Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures.

Majors

Ehrhart

1993

Arterioscler Thromb

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To determine the effects of an intact extracellular matrix on collagen synthesis, arterial smooth muscle cells (SMCs) were plated sparsely on a cell-free, SMC-derived matrix and examined the following day. Collagen synthesis during a 5-hour incubation by cells on the matrix was reduced to 67% of the control values obtained from cultures on plastic. Total protein synthesis was unaffected. Treatment of the matrix with heparitinase to remove basic fibroblast growth factor (bFGF) before seeding the SMCs abolished the inhibitory effect of the matrix on collagen synthesis. The inhibitory effect was also eliminated by treating the matrix with a neutralizing polyclonal antibody directed against bFGF. Collagen synthesis by SMC cultures grown in wells coated with purified bFGF was only 61% that of control cultures, whereas total protein synthesis remained unchanged. Slot-blot analysis revealed that the relative message level for orl(III) procollagen was reduced in cultures grown on the preexisting matrix or on plastic precoated with bFGF, whereas the al(I) procollagen message was unaffected. These results demonstrate the ability of the extracellular matrix to modulate the synthesis of collagen by arterial SMCs and indicate that bFGF in the matrix is responsible for these effects. (Arteriosclerosis and Thrombosis 1993; 13:680-686) KEY WORDS • collagen • smooth muscle cells • basic fibroblast growth factor • extracellular matrixC ells in vivo are in contact with an insoluble matrix composed of collagens, elastin, proteoglycans, noncollagenous glycoproteins, and a variety of adherent nonstructural components including growth factors. This extracellular matrix has the capacity to modulate the behavior and phenotype of cells both in vivo and in vitro. Several types of cultured cells have been shown to respond to the substratum onto which they are plated by altering their adhesion, growth, morphology, and protein synthesis patterns.Numerous studies have shown that preformed matrices, when used as a substratum for cultured cells, may have significant effects on cell proliferation.1 " 3 In addition, the substratum on which cells are plated can have specific effects on the synthesis of extracellular matrix components.4 " 6 Previous studies in our laboratory have demonstrated that preconfluent smooth muscle cells (SMCs) grown on fibronectin synthesize significantly less collagen and fibronectin than control SMCs, whereas noncollagenous protein synthesis was unaltered. 7 In contrast, both collagen and fibronectin syn-

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Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth μscle cells in culture

Cited by 26 publications

References 34 publications

Measurement of protein metabolism in epidermis and dermis

Measurement of protein metabolism in epidermis and dermis

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running

Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures.

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