Age-related changes in enzyme activities, protein content and lipid composition of rat kidney brush-border membrane.

Age-related changes in enzyme activities, protein electrophoretic patterns and lipid composition of kidney-brush-border membranes were studied in 10-20- and 30-month-old male and female Wistar rats. Polyacrylamide gel electrophoresis of membrane proteins revealed very little changes with increasing age, whether males or females were considered. The Km of three hydrolases - maltase, L-aminopeptidase and alkaline phosphatase - were not affected by age while the Vmax of maltase and alkaline phosphatase, but not of L-aminopeptidase, decreased from 10 to 30 months. The phospholipid to protein ratio which remained constant between 10 and 20 months, rose in both sexes from 20 to 30 months. In males, the cholesterol content of the membrane increased faster than that of phospholipid and the cholesterol over phospholipid ratio was then greater at 30 months than at 10 months, while in females this ratio remained unchanged in the course of aging. The fatty acid composition of the brush-border remained more or less constant with age in female rat whereas in the male, a 10% decrease in the proportion of arachidonic acid from 10 to 30 months was responsible for a lower unsaturation index.

Evidence for proteic water pathways in the luminal membrane of kidney proximal tubule.

The osmotic permeability of the apical membrane of proximal tubule cells was studied on rat brush-border membrane vesicles by following their rate of shrinkage with a stopped-flow device coupled to light transmission recording. The mercuric sulfhydryl reagent para-chloromercuribenzenesulfonic acid (PCMBS) reduced the water permeability of the membrane, in a time- and dose-dependent manner, to 35% of the control value. Mercuric chloride was a more potent inhibitor and decreased the osmotic water permeability of the brush-border membrane to 15% of the control. This inhibition was reversed by an excess of cysteine, while cysteine per se did not modify the rate of vesicle shrinkage. These results suggest that most of the osmotic water movements across kidney brush-border membranes are through polar pathways which involve the integrity of the membrane proteins.

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles.

Solute reflection coefficients, sigma i, of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for sigma NaCl and 0.56 for sigma KCl when compared with 0.92 for sigma mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought sigma NaCl and sigma KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index.

Solute reflection coefficients sigma of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of sigma. We determined sigma NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone (Mr 40,000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a sigma NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again sigma NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule.

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.

Aging affects choroidal proteins involved in CSF production in Sprague-Dawley rats.

Aging is currently associated with progressive declines of cerebral functions. From these, a decreased resistance to dehydration suggested alteration in choroidal control of brain homeostasis and reduced cerebrospinal fluid (CSF) production in old subjects. In the present study, choroid plexuses of 20-month old Sprague-Dawley rats were compared with those of 3- and 10-month old rats. Using ultrastructure analysis and immunodetection of ezrin, a protein associating cytoskeleton to membranes, we showed that progressive loss of microvilli and strong decrease in apical ezrin are evident in 20-month old rats. Using immunolabeling and confocal microscopy, we found reduction in expression of two choroidal proteins, carbonic anhydrase II and aquaporin 1, involved in CSF secretion. In addition, we confirmed previous studies indicating that choroidal Na,K-ATPase decreased with age. In situ hybridization analyses showed that mRNA levels for Na,K-ATPase and aquaporin 1 were significantly lowered in choroid plexus of old rats. These findings are consistent with a reduced secretory activity in choroid plexus and suggest that massive disorders could affect choroidal CSF production in aged rats.

Homologous and heterologous phosphorylation of the vasopressin V1a receptor.

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.

Angiotensin II potentiates vasopressin-dependent cAMP accumulation in CHO transfected cells. Mechanisms of cross-talk between AT1A and V2 receptors.

The V2 vasopressin and the AT1A angiotensin II receptors are respectively coupled to the adenylyl cyclase and the phosphoinositide pathways. The cross-talk between these two receptors and their transduction pathways were investigated in CHO cells transfected with cDNA of both AT1A and V2 receptors. In these cells, angiotensin II induced an increase in intracellular calcium, and vasopressin a rise in intracellular cAMP accumulation. The simultaneous addition of angiotensin II and vasopressin potentiated the production of cAMP by the V2 receptor. This potentiation was dose-dependent and, at a concentration of 10(-7) M angiotensin II, the accumulation of cAMP was 4-fold greater than that induced by 10(-7) M vasopressin alone. Such cross-talk occurred in the presence and absence of cyclic nucleotide phosphodiesterase inhibitors, indicating that inhibition of phosphodiesterase activity was not the principal cause of potentiation. This was confirmed by the absence of calcium-inhibitable isoforms of phosphodiesterases in CHO cells. The addition of angiotensin II to forskolin, which stimulates the adenylyl cyclase, did not modify the production of cAMP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), partially mimicked, and staurosporine, an inhibitor of PKC, partially inhibited the effect of angiotensin II on vasopressin. Chelation of intracellular calcium with BAPTA-AM markedly reduced the potentiation of V2 receptor by angiotensin II. However, increase in intracellular calcium with thapsigargin did not modify the cAMP accumulation induced by vasopressin. It was concluded that, in CHO cells, activation of the AT1A receptor by angiotensin II potentiates the V2 receptor through activation of protein kinase C in the presence of intracellular calcium at a step located between the receptor and the adenylyl cyclase.

Quantitative analysis of the Purkinje cell population during extreme ageing in the cerebellum of the Wistar/Louvain rat.

The loss of neurons is viewed as one of several causes of the deterioration of neural function during ageing. However, the existing experimental evidence for an age-related decrease in the neuronal number may be misinterpreted due to the way the cells are counted and to the interference of unsuspected degenerative pathology of the animals studied. To reinvestigate this question we have quantified an easily identifiable population of neurons, the cerebellar Purkinje cells, in very old but healthy rats. The number of Purkinje cells in the cerebellum was assessed in two populations of rats: control (10 months) and old (42 months) rats from the Wistar/Louvain strain. In both groups, paraffin-embedded brains were cut serially in the sagittal plane. Purkinje cells were counted every 15 or 22 sections under the light microscope at a magnification of 1250 x. The raw value of cell counts were corrected according to the method of Hendry (21) in order to avoid the overestimation due to splitting of the nucleus during sectioning. The latero-lateral extent of the cerebellar cortex, obtained by multiplying the thickness of the section by the number of sections in which Purkinje cells were counted, was not statistically different (mean +/- standard deviation): 12.8 +/- 1.16 mm (n = 6) for the control rats and 12.0 +/- 1.02 mm for the old animals (n = 8) (Student's t-test, p = 0.18). The corrected number of the Purkinje cells (mean +/- standard deviation) was 330,350 +/- 35,448 cells (n = 6) for the control animals and 299,019 +/- 50,223 (n = 8) cells for the old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of protein kinase C and carboxyl-terminal region in acute desensitization of vasopressin V1a receptor.

The role of protein kinase C activation and carboxyl-terminal region in rapid desensitization of the vasopressin V1a receptor was investigated in Xenopus oocytes. Preincubation of the oocytes with vasopressin or with the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), or direct injection of active protein kinase C, all blunted the calcium response of the V1a receptor. Truncation of the 51 terminal amino acids (S374STOP) modified neither the intracellular calcium response to vasopressin nor its desensitization by vasopressin or OAG. These data suggest that desensitization of the V1a receptor is mediated by PKC activation and that its carboxyl-terminal domain is not required for signal transduction and rapid desensitization.

Role of the carboxyl-terminal region, di-leucine motif and cysteine residues in signalling and internalization of vasopressin V1a receptor.

The structural requirements for internalization and signalling of the vasopressin V1a receptor were investigated in stably transfected HEK-293 cells. Removal of the 51 C-terminal amino acids did not affect vasopressin binding, calcium signalling, heterologous desensitization or internalization of the receptor. Deletion of 14 additional amino acids reduced vasopressin-dependent calcium increase and impaired receptor internalization. Substitution of cysteines 371-372 did not affect intracellular signalling, but decreased endocytosis by 26%. Substitution of the 361-362 leucine by alanine residues reduced by 56% V1a receptor sequestration without affecting calcium signalling. These results indicate that di-cysteine and mostly di-leucine motifs present in the C-terminal region of the V1a receptor are involved in its internalization.

Plasma vasopressin and cortical nephron function in aging rats.

The role of vasopressin and Henle's loop transport in age-related polyuria and decrease in urine osmolality was investigated in female WAG/Rij rats free of kidney disease. In these animals, urine osmolality dropped from 2000 mosmol/kg H2O to 1000-1200 mosmol/kg H2O between 10 and 30 months, and urinary volume increased in proportion. Vasopressin concentration measured in plasma withdrawn from conscious, unrestrained, chronically catheterized rats was not significantly different in 10, 20 and 30-month-old animals (mean values 2.5 +/- 0.7, 2.2 +/- 0.2 and 2.0 +/- 0.3 pg/ml (n = 8), respectively). This suggests an impaired responsiveness of old kidney to antidiuretic hormone. The possible involvement of Henle's loop in this defect was studied by micropuncture. Paired collections of tubular fluid were done in the early distal and late proximal convolutions of the same cortical nephrons. Single nephron filtration rates did not significantly differ with age. Tubular fluid osmolalities in the early distal convolution were 165 +/- 13, 178 +/- 9 and 160 +/- 11 (n = 14) mosmol/kg H2O in 10-, 20- and 30-month-old rats, indicating similar diluting capacity of the cortical thick ascending limb. The amount of sodium transported from lumen to peritubular space by Henle's loop was also unchanged with age as were water, calcium, magnesium and potassium reabsorptions. These data indicate that the age-related decrease in urine osmolality is not related to either a significant reduced vasopressin plasma concentration or an increased single glomerular filtration rate or a reduced transport capacity of Henle's loop of the cortical nephron. Rather they suggest an impaired response to vasopressin of other segments of the nephron that is, the medullary thick ascending limb of Henle's loop and/or the collecting duct.

Effect of aging on zinc and histidine transport across rat intestinal brush-border membranes.

The effects of aging on intestinal absorption of zinc and L-histidine were investigated in adult (10-month-old) and senescent (30-month-old) Wistar rats' brush-border membrane vesicles isolated from jejunum and ileum. Kinetic parameters of the zinc transport by the jejunal brush-border membrane were Jmax = 126 +/- 24 nmol.min-1.mg-1 protein and Km = 490 +/- 126 microM (10-month-old rats, n = 7). The transport of zinc was the same in the jejunum and the ileum of adult animals. In senescent rats, the zinc uptake was significantly lower in the distal part of the intestine than in the proximal one. A comparison of zinc uptake in 10- and 30-month-old rats showed that the transport capacity of the jejunum did not change with age but the ileal transport capacity decreased by 50%. This reduced uptake was associated with an increased cholesterol content of the brush-border membrane. The major site of L-histidine absorption was the jejunum, in both the 10- and 30-month-old animals. L-Histidine was co-transported with Na+. The kinetic parameters of the L-histidine carrier in the presence of Na+ were Jmax = 6.5 +/- 1.0 nmol.min-1.mg-1 protein and Km = 190 +/- 29 microM in the jejunum of 10-month-old rats (n = 12). Increasing the extra-vesicular concentration of zinc (0 --> 1 mM) reduced the uptake of L-histidine, and conversely increasing the concentration of L-histidine (0 --> 1 mM) reduced that of zinc: there was no evidence of transport of a complexed form [zinc-L-histidine] in brush-border membranes of the small intestine. During aging, the transport capacity of L-histidine by the jejunum decreased, whereas the ileal transport capacity was conserved. The modifications of absorptive capacity for zinc and L-histidine at the membrane level (loss of ileal function for zinc, and loss of jejunal function for amino acid) indicate that the normal aging of intestinal epithelial cells cannot be regarded as a decline in the overall transport of nutriments but as a combination of highly specific modifications of the various transport systems.

Glomerular capillary network of cortical nephrons is reduced in male but not in female aging rats.

The gender differences in the age-related changes of glomerular structures were determined in 10- and 30-month-old rats. In adult animals, glomerular volume, urinary space, capillary lumen area and mesangial domains of deep and superficial nephrons were larger in males than in females. Glomerular hypertrophy was evidenced with age in both males and females. This hypertrophy was greater in female (+70%) than in male (+20%) rats. Age-related hypertrophy concerned equally the urinary space and the glomerular tuft. The mesangial domain, however, increased more markedly than glomerular volume (+400%). As a result, the ratio of mesangial domain to glomerular section area was more than doubled between 10 and 30 months. In females, the age-related renal hypertrophy was associated with a constant total capillary lumen area in cortical nephrons. In contrast, the total capillary lumen area of male rats was reduced by 20% in superficial glomeruli and by 36% in deep glomeruli between 10 and 30 months. These morphological changes are in good agreement with the maintained glomerular filtration rate reported in old female rats and the decrease in renal blood flow and filtration rate reported in male rats. They suggest that the aging process does not similarly affect the vascular system of the kidney of male and female rats, although their mean blood pressure was comparable.

Glycation of albumin with aging and diabetes in rats: changes in its renal handling.

Albumin glycation was investigated in old rats to elucidate the link between the preferential excretion of glycated albumin and age-related microalbuminuria. Postprandial blood glucose and the glycated albumin in the serum and urine of 3-, 10- and 30-month-old Wistar rats and in streptozotocin diabetic rats were determined. Blood glucose increased from 1.46 +/- 0.046 g l-1 in 3-month-old rats to 2.08 +/- 0.06 (10 months) and 1.75 +/- 0.23 (30 months) (P < 0.05). Albumin glycation level in the serum increased from 0.79 +/- 0.07 nmol HCHO/nmol albumin (3 months) to 1.41 +/- 0.14 (10 months) and 1.73 +/- 0.21 (30 months) (P < 0.05); urinary level increased from 1.63 +/- 0.39 nmol HCHO/nmol albumin (3 months) to 2.92 +/- 0.57 (10 months) and 2.39 +/- 0.36 (30 months) (P < 0.01). The percent glycated albumin in serum rose from 3.33 +/- 0.64 to 6.81 +/- 0.63 and 6.99 +/- 1.79% of total albumin (P < 0.05), whereas the urine percentage decreased from 12.81 +/- 3.97 to 12.64 +/- 2.87 and 2.63 +/- 0.97% (P < 0.05) in 3-, 10- and 30-month-old rats, respectively. Editing decreased with aging from 4.28 +/- 0.83 (3 months) to 1.84 +/- 0.32 (10 months) and 0.52 +/- 0.14 (30 months) (P < 0.01). Editing in microproteinuric diabetic rats was lower (0.95 +/- 0.08) than in 3-month-old control rats (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular signaling, AGE accumulation and gene expression in hepatocytes of lean aging rats fed ad libitum or food-restricted.

The effects of food restriction on liver glucagon and vasopressin V1a receptors, on AGE accumulation and on gene expression were investigated in 10- and 30-month-old WAG/Rij female rats fed ad libitum or chronically food-restricted by 30%. The age-related increase in glucagon and vasopressin V1a receptor density, as well as the rise in glucagon-induced cAMP generation was prevented by the restriction. AGE accumulation, characteristic of the aging process, was normalized in food-restricted animals. Gene expression determined with rat Atlas cDNA Expression Arrays containing 1176 cDNA indicates that a few genes exhibited a greater than twofold change in mRNA ratios with age. Most down-regulated genes were related to oxidative metabolism of lipids, and most of the up-regulated genes were concerned with the cell cycle and transcription factors. Chronic food restriction partially prevents these changes in gene expression and induces up- and down-regulation of several mRNAs which are not modified with age in ad libitum fed rats.

Glucagon and vasopressin V1a receptor signaling in hepatocytes from aging rats.

Glucose tolerance is reduced with age. The relationship between this change in glucose homeostasis and signaling of glucagon and vasopressin V1a receptors was investigated in hepatocytes isolated from 10- and 30-month-old female WAG/Rij rats. Binding capacity of hepatocytes for 125I glucagon and 3H vasopressin increased 2- and 1.8-fold, respectively, between 10 and 30 months. Intracellular cAMP accumulation induced by glucagon was 40% greater in hepatocytes of aging rats than of adults, although EC(50) were similar in the two groups. Conversely, phosphodiesterases activity and nucleotides leakage out of the cells were unchanged with age. The rise in intracellular calcium consecutive to the stimulation of V1a receptor was comparable in adult and senescent animals. Finally, glucose release by hepatocyte suspensions was greater in senescent than in adult animals in absence as in presence of glucagon. These experiments suggest that increase in glucagon receptor expression and cAMP generation would contribute to the impaired glucose tolerance characteristic of the aging process.

Renin gene expression in the aging kidney: effect of sodium restriction.

The activity of the renin-angiotensin system as well as the ability of the kidney to retain sodium following salt restriction are reduced with age. The relationship between these age-related changes in renal function and the renin gene expression was presently investigated. The concentrations of renin and its mRNA were measured in kidney of 10- and 30-month-old control female WAG/Rij rats and of animals which were salt restricted for 4 days. In the senescent rats, the kidney renin concentration, like the plasma concentration of angiotensin II, was half that in adult rats. The intrarenal content of renin mRNA did not differ between 10- and 30-month-old animals, suggesting that the transcriptional rate of the renin gene is unchanged with age. During the early phase of adaptation to sodium depletion, the systemic angiotensin II concentration was not modified in either age groups. Four-days salt restriction did not significantly change the renal storage of renin. In contrast, this short term salt restriction induced a 2.3-fold increase in the renin mRNA in adult kidney, and a 1.9-fold increase in the senescent kidney. These data suggest that the age-related decrease in renal concentration of renin is linked to a modification in the rate of translation of renin mRNA, or to an alteration in the protein maturation. The difference in adaptation to the early phase of salt restriction with age should not be linked to changes in renin gene transcription, but more likely to a change in the tissue response to the local renin-angiotensin system.

Myocardial effect of converting enzyme inhibition in hypertensive and normotensive rats.

The effects of converting enzyme inhibition on the cardiac mass, isomyosins polymorphism, and collagen network in the left ventricle have been studied in renovascular, hypertensive, spontaneously hypertensive, and normotensive rats. The isoenzyme profile of left ventricular myosins was used as an indirect marker of the intrinsic property of contractility, whereas the collagen network, measured by a morphometric method, represented an indirect structural marker of the arrhythmogenic risk. One-clip, two-kidney renovascular hypertension was associated with cardiac hypertrophy, a shift in the isomyosin profile, and accumulation of collagen within the left ventricular myocardium. In this renin-angiotensin-dependent model, one month of treatment with converting enzyme inhibitor normalized blood pressure and consistently reversed cardiac hypertrophy and the isomyosin profile. Converting enzyme inhibitor treatment of 12-week-old spontaneously hypertensive rats for three months significantly decreased blood pressure but did not completely normalize it. The increase in cardiac mass observed in spontaneously hypertensive rats was not reversed by this short treatment. Nevertheless, the percentage of the V1 form of myosin increased slightly after treatment, and the collagen content of the left ventricle was considerably decreased. Converting enzyme inhibition did not decrease blood pressure in DOCA-salt hypertension, and no changes were observed in cardiac hypertrophy, isomyosin profile, or the collagen network. The cardiac hypertrophy that occurs with aging in normotensive rats was associated with a significant shift in isomyosin profile and a large accumulation of collagen. Thus, aging mimics several of the quantitative and qualitative changes in the left ventricular protein profile observed in hypertension. In young normotensive rats, converting enzyme inhibition significantly decreased blood pressure and left ventricular mass, increased the percentage of V1 isomyosin, and prevented the accumulation of collagen. In one-year-old normotensive rats, treatment for six months with converting enzyme inhibitor decreased blood pressure, decreased cardiac mass, and prevented the accumulation of collagen; the isomyosin profile was not modified. Converting enzyme inhibition, by acting on cardiac afterload, can bring about quantitative and qualitative changes in the cardiac proteins of both hypertensive and normotensive rats.

Effects of chronic and acute aminoguanidine treatment on tail artery vasomotion in ageing rats.

1. This study was designed to evaluate the effects of aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), on the reactivity and intracellular calcium ([Ca(2+)](i)) mobilization induced by noradrenaline in the perfused tail artery from aged WAG/Rij rats. Global mean internal diameter was 350+/-15 microns and wall thickness 161+/-3 microns. The influence of the endothelium on these responses was also analysed. The intracellular dye fura-2 for [Ca(2+)](i) measurements was used. 2. Noradrenaline-induced vasoconstriction decreased progressively from 3 to 20 and 30 months. Removal of the endothelium attenuated vasoconstriction in 20 and 30 month-old rats (P<0.05) but not in young rats. 3. Chronic administration of aminoguanidine (50 mg kg(-1) day(-1), p.o.) to WAG/Rij rats from 20 to 30 months enhanced (P<0. 01) the [Ca(2+)](i)-sensitivity of noradrenaline-induced vasoconstriction. 4. Aminoguanidine (300 microM) in vitro significantly shifted the concentration-vasoconstriction curve to noradrenaline to the left (P<0.01) in denuded vessels from both 20 and 30 month-old rats. The acute inhibitory effect of aminoguanidine was also observed after chronic aminoguanidine treatment. Aminoguanidine failed to modify vasoconstriction in the presence of the endothelium. 5. Acute aminoguanidine (300 microM) treatment did not modify vasoconstriction induced by noradrenaline in young rats. 6. Quantification of iNOS mRNA expression in tail arteries from 3 and 20 month-old WAG/Rij rats showed that expression was enhanced (x2.1, P<0.01) with age. 7. These results suggest that an inflammatory process develops in the media of the rat tail artery with age and that the subsequent increase in non-endothelial iNOS activity attenuates noradrenaline-induced vasoconstriction.