WOUND HEALING AND COLLAGEN FORMATION : III. A Quantitative Radioautographic Study of the Utilization of Proline-H3 in Wounds from Normal and Scorbutic Guinea Pigs

The sequence of incorporation and utilization of tritium-labeled proline has been examined in healing wounds from normal and scorbutic guinea pigs. Linear incisions in the skin of the animals were allowed to heal for 7 days. Each animal was given proline-H³, and the wounds were excised 30 minutes, 1 and 4 hours, 1, 3 and 7 days after proline administration. The tissues were fixed in osmium tetroxide, fixed again in neutral buffered formalin, embedded in epoxy resin, and sectioned at 1 micron thickness. The sections were coated with nuclear track emulsion, exposed, developed, and stained. The results of grain counts were quantitated as the number of counts per unit area overlying cells, fibers, etc. In both groups the proline reaches a maximum over the fibroblasts within 4 hours and subsequently disappears from the cells. Concomitantly, the proline reaches a maximum over the collagen (in normal animals) and extracellular fibrillar material (in scorbutic animals) by 4 hours, where it remains. The modified technique of radioautography used in this study allows not only resolution of approximately 1 micron, but also minimal background, decreased artifact, and a clear separation of the randomly situated elements within the wounds so that grain counting is facilitated. The results correlated with previous electron microscopic studies are consistent with the utilization of proline by the fibroblasts and its incorporation into collagen (in normal animals) and into the extracellular, fibrillar, non-collagenous material seen in scorbutic animals.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

WOUND HEALING AND COLLAGEN FORMATION : IV. Distortion of Ribosomal Patterns of Fibroblasts in Scurvy

The changes in scorbutic wounds following the administration of ascorbic acid have been investigated using the techniques of electron microscopy, histochemistry, and autoradioggraphy. Particular attention has been paid to the changes seen in the endoplasmic reticulum of the fibroblasts and to the identity of the extracellular filamentous material characteristic of scorbutic wounds. Seven-day-old wounds in scorbutic guinea pigs were examined prior to and from one to 72 hours following the administration of vitamin C. Fibroblasts from wounds of normal animals demonstrate a characteristic configuration of the ribosomes of the endoplasmic reticulum which is suggested to be analogous to polyribosomes described in cells synthesizing protein such as the reticulocyte. Tangential views of the membranes of the ergastoplasm show the ribosomes to be grouped in paired rows which take both straight and curved paths. This configuration is lost in scurvy and can be seen to begin to reappear as early as 4 hours after giving ascorbic acid. With increasing time, the morphology of the ribosomal aggregates approximates that seen in normal cells, so that by 24 hours their reorientation is complete. It is suggested that one of the disturbances in scurvy may relate to an alteration either in messenger RNA, in the ability of the ribosomes to relate to the messenger, or in the membranes of the ergastoplasm. In addition, the lack of formation of hydroxyamino acids necessary for completing collagen synthesis may be related to the architecture of the ribosomal aggregates. Extracellular collagen fibrils appear concomitant with the restoration of ribosomal and ergastoplasmic morphology as early as 12 hours after administration of ascorbic acid, with complete disappearance of the scorbutic extracellular material within 24 hours. Observations of this scorbutic material do not support the concept that it is a collagen precursor.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

WOUND HEALING AND COLLAGEN FORMATION : I. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the Electron Microscope

The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Complement component C1q is sensitive to acute vitamin C defficiency in guinea pigs

In acutely scorbutic guinea pigs, where interstitial collagen synthesis is markedly impaired, there was no significant reduction in total complement component C1 activity measured by a functional assay, and no significant reduction in the ratio of protein-bound hydroxyproline to protein-bound proline or to total serum protein, in comparison with pair-fed controls. There was a moderate increase in non-protein-bound hydroxyproline in the serum of the deficient animals.These result suggest that component C1q is largely resistant to the effects of severe acute scurvy, adn that some hydroxyproline-containing proteins may respond differently others, during vitamin C deficiency.     

Histomorphological study of wound regeneration in animals irradiated for a long time with low-intensity microwaves]

Experimental study on 80 guinea pigs showed that in the animals exposed for a long time to the action of microwabes of low intensity the linear skin wounds healed by primary intension much more rapidly than in the non-irradiated animals. The postoperative scar was much more reliable in the irradiated animals. On the third day the irradiated animals were found to have a more intensive regeneration of the epithelium in the inflicted wound, and an intensive development of the granular tissue in the inferior portions. Later, an aceelerated synthesis of proteins, including that of collagen in the wound was observed; there was also a marked process of maturation of the granular tissue and formation of the intercellular substance. On the 7th-9th days fresh connective tissue (rich in fibroblasts with young fuchsinophilous collagen fibers) replaced the granular tissue; it was more mature than in the non-irradiated animals.     

Structural and mechanical factors influencing infarct scar collagen organization

Although large collagen fibers in myocardial infarct scar are highly organized, little is known about mechanisms controlling this organization. The preexisting extracellular matrix may act as a scaffold along which fibroblasts migrate. Conversely, deformation within the ischemic area could guide fibroblasts so new collagen is oriented to counteract the stretch. To investigate these potential mechanisms, we infarcted three groups of pigs. Group 1 served as infarct controls. Group 2 had the endocardium slit longitudinally to alter local systolic deformation. Group 3 had a plug sectioned from ischemic tissue and rotated 90 degrees. The slit altered systolic deformation in the infarcted tissue, changing circumferential strain from expansion to compression and increasing radial strain and shears and the variability of collagen fiber angles but not the mean angle. In the plug pigs, when deformation, matrix orientation, and continuity are altered in the infarct area, the result is complete disarray in the organization of collagen within the infarct scar.     

Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.     

Coordinate regulation of collagen and proteoglycan synthesis in costal cartilage of scorbutic and acutely fasted, vitamin C-supplemented guinea pigs

The effects of ascorbic acid deficiency and acute fasting (with ascorbate supplementation) on the synthesis of collagen and proteoglycan in costal cartilages from young guinea pigs was determined by in vitro labeling of these components with radioactive proline and sulfate, respectively. Both parameters were coordinately decreased by the second week on a vitamin C-free diet, with a continued decline to 20-30% of control values by the fourth week. These effects were quite specific, since incorporation of proline into noncollagenous protein was reduced by only 30% after 4 weeks on the deficient diet. The time course of the decrease in collagen and proteoglycan synthesis paralleled the loss of body weight induced by ascorbate deficiency. Hydroxylation of proline in collagen synthesized by scorbutic costal cartilage was reduced to about 60% of normal relatively early, and remained at that level thereafter. Neither collagen nor proteoglycan synthesis was returned to normal by the addition of ascorbate (0.2 mM) to cartilage in vitro. Administration of a single dose of ascorbate to scorbutic guinea pigs increased liver ascorbate and restored proline hydroxylation to normal levels by 24 h, but failed to increase the synthesis of collagen or proteoglycan. Synthesis of both extracellular matrix components was restored to control levels after four daily doses of ascorbate. A 96-h total fast, with ascorbate supplementation, produced rates of weight loss and decreases in the synthesis of these two components similar to those produced by acute scurvy. There was a linear correlation between changes in collagen and proteoglycan synthesis and changes in body weight during acute fasting, scurvy, and its reversal. These results suggest that it is the fasting state induced by ascorbate deficiency, rather than a direct action of the vitamin in either of these two biosynthetic pathways, which is the primary regulatory factor.     

Alterations in the activities of intestinal enzymes in vitamin-C-deficient guinea pigs

The effect of vitamin C deficiency on various enzymes of the intestinal epithelium has been studied in guinea pigs. Brush border sucrase and alkaline phosphatase activities were considerably enhanced (p less than 0.001), but leucine aminopeptidase levels were reduced in scorbutic animals compared to the control group. There was essentially no change in the activity of maltase under these conditions. Kinetic studies with sucrase and alkaline phosphatase in control and scorbutic animals revealed that augmentation of the enzyme activities in scurvy is due to enhanced enzyme contents. Lactate dehydrogenase, succinate dehydrogenase, glucose-6-phosphatase and Mg+2 ATPase also exhibited reduced activities in the intestine of vitamin-C-deficient animals. Observed alterations in the activities of intestinal enzymes in scurvy were restored to control levels upon feeding of vitamin C to scorbutic guinea pigs.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.     

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine

The effects of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and kidney carnitine levels were not affected by the deficiency, but scorbutic animals had 50% less carnitine in heart and skeletal muscle than control animals. Labeled carnitine precursors, 6-N-tri-methyl-L-lysine and 4-N-trimethylaminobutyrate, both of which require ascorbate for their enzymatic hydroxylation, were injected into the vena cava of control, pair-fed and scorbutic animals. The distribution of isotope in compounds present in the liver and kidney after 1 h was determined. The uptake of trimethyllysine by the liver was less than 2% in 1 h, while the kidney took up approx. 20% of the 14C. Control and pair-fed animals converted trimethyllysine to kidney trimethylaminobutyrate 8--10 times as well as did scorbutic animals. Trimethylaminobutyrate hydroxylase, present in the liver but almost absent from the kidney, converted nearly all of substrate taken up by the liver to carnitine in both the scorbutic and control animals.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.     

Airway responsiveness and prostaglandin generation in scorbutic guinea pigs

Airway responsiveness to histamine aerosol and lung prostaglandin generation were investigated in normal, partially vitamin C deficient and scorbutic guinea pigs. The ascorbic acid content of the lung expressed as microgram/100 mg wet weight lung parenchyma decreased from 22.1 +/- 1.8 (mean +/- SE) in the control group to 9.0 +/- 1.4 and 1.8 +/- 0.4 in tissues from partially ascorbic acid deficient and scorbutic animals, respectively. Guinea pigs on low and ascorbic acid deficient diets developed significant airway hyperresponsiveness to histamine aerosol after 3 and 4 weeks. Indomethacin (30 mg/Kg, i.p.) further increased the airway hyperresponsiveness in scorbutic animals but was without effect in control animals. Prostaglandin generation from different parts of the lung was significantly changed by the diets. However, airway hyperresponsiveness was not directly attributable to altered prostanoid generation. Scorbutic conditions did not alter the electrophysiological characteristics of airway smooth muscle namely, resting membrane potential and electrogenic sodium pump activity. In summary, ascorbic acid deficiency causes airway hyperresponsiveness to histamine in guinea pigs. This alteration seems not to be related to an altered prostaglandin generation by the lung or to the electrophysiological properties of airway smooth muscle.