Developmental changes in collagen and elastin biosynthesis in the porcine aorta

Elastin and collagen are the principal scleroproteins of the aortic wall, and they largely determine its physical and mechanical properties. During perinatal development of the aorta, elastin and collagen accumulate rapidly, being present as inverse gradients by the time of birth. Elastin is most prevalent in the thoracic aorta, decreasing distally, while collagen shows the opposite trend. The present studies have determined the relative and absolute rates of collagen and elastin synthesis in the porcine aorta between 60 days of fetal development (mid-gestation) and 110 days after birth. Although there was measurable elastin synthesis in the upper thoracic aorta at the earliest time evaluated, there was a fourfold increase in relative elastin synthesis (from 4 to 16% of total protein synthesis) between 60 fetal days and birth. Elastin synthesis was maximal in successively distal segments between 1 and 3 weeks after birth. Relative collagen synthesis progressively increased in distal aortic regions between 90 fetal days and 60 days postpartum. Greater than twofold increases over thoracic levels were measured. Both elastin and collagen synthesis largely subsided by 110 days of development. When expressed as absolute rates of protein synthesis, these scleroproteins were maximally expressed in the first 3 postnatal weeks. Elastin mRNA levels were determined with a cloned sheep gene fragment by molecular hybridization. Gradients of elastin message were present at 60 fetal days and at 4 and 14 days after birth, elastin mRNA levels being maximal in the upper thoracic aorta at 14 days after birth. The differentiation of the aortic wall thus follows discrete patterns of phenotypic change which may be coupled to the rheologic stresses accompanying development of the circulatory system.     

Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle.

Application of cyclic stretch (10% at 1 hertz) to vascular smooth muscle cells (SMC) increased L-arginine uptake and this was associated with a specific increase in cationic amino acid transporter-2 (CAT-2) mRNA. In addition, cyclic stretch stimulated L-arginine metabolism by inducing arginase I mRNA and arginase activity. In contrast, cyclic stretch inhibited the catabolism of L-arginine to nitric oxide (NO) by blocking inducible NO synthase expression. Exposure of SMC to cyclic stretch markedly increased the capacity of SMC to generate L-proline from L-arginine while inhibiting the formation of polyamines. The stretch-mediated increase in L-proline production was reversed by methyl-L-arginine, a competitive inhibitor of L-arginine transport, by hydroxy-L-arginine, an arginase inhibitor, or by the ornithine aminotransferase inhibitor L-canaline. Finally, cyclic stretch stimulated collagen synthesis and the accumulation of type I collagen, which was inhibited by L-canaline. These results demonstrate that cyclic stretch coordinately stimulates L-proline synthesis by regulating the genes that modulate the transport and metabolism of L-arginine. In addition, they show that stretch-stimulated collagen production is dependent on L-proline formation. The ability of hemodynamic forces to up-regulate L-arginine transport and direct its metabolism to L-proline may play an important role in stabilizing vascular lesions by promoting SMC collagen synthesis.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

In vitro PGI2 synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI2 produced were shown as 6-keto-PGF1 alpha per microgram of aortic tissue DNA instead of per mg wet weight. We also investigated PGI2 synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima. Basal PGI2 production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI2 production by atherosclerotic aorta was also significantly higher than that of controls. PGI2 producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI2 production than the medial layer. Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI2 than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI2 production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Response of aorta connective tissue matrix to injury caused by vassopressin-induced hypertension or hypercholesterolemia.

The aim of the study was to investigate the effect of two various atherogenic stimuli (vasopressin-induced hypertension or hypercholesterolemia) on the collagen and glycosaminoglycan (GAG) content in the internal or external part of both thoracic and abdominal aorta, which are differently susceptible to atherosclerosis. Experimental rabbits were divided into four groups: controls, animals injected with physiological saline or vasopressin at the dose of 1 IU/kg from the 1 st to the 25 th day of experiment, respectively. The animals from group 4 were maintained on food, containing 0.25% cholesterol. Only in the vasopressin-treated group, the systolic blood pressure was elevated from 110 mmHg at the beginning, to 166 mmHg at the end of the study. After 14 weeks the aorta was dissected into internal and external parts. GAG fractions were separated and estimated as uronic acids. Collagen was evaluated as the hydroxyproline content in the tissue. Augmented total GAG and heparan sulphate (HS) level, plus no changes in the collagen content were seen in the internal part of the thoracic aorta in rabbits with hypercholesterolemia or hypertension. In the hypertensive animals, the changes were extended to the external part of the aorta and, additionally, comprised the elevation of the chondroitin-4 sulphate (C-4S) content. The two atherogenic stimuli increased the collagen level with no elevation of the GAG content in the abdominal aorta. A convergent effect of the injury, caused by hypertension or hypercholesterolemia on the collagen, total GAG and HS content was shown in the respective parts of the rabbit aortas. The common GAG, increased in the thoracic aorta, stand for the HS, in both hypertensive and hypercholesterolemic rabbits. As the sensitivity to atherosclerosis development in different segments of the aorta varies, they express various responses of the connective tissue matrix to injuries, caused by hypertension or hypercholesterolemia.     

Glycosylated collagen

Collagen was separated from segments of thoracic aorta excised from normal and streptozotocin-induced diabetic rats. The extent of collagen glycosylation was determined using a colorimetric chemical procedure specific for the detection of ketoamine-linked hexoses in proteins. Diabetic rats exhibited a significant increase in glycosylated collagen as compared to normal animals. Glycosylated collagen may contribute to the development of diabetic vascular complications.     

Modulation of DNA synthesis in aortic smooth muscle cells by dinitrotoluenes

Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced ³H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis     

Differential PDGF secretion by graft and aortic SMC in response to oxidized LDL

Smooth muscle cells (SMC) from prosthetic vascular grafts constitutively secrete higher levels of platelet-derived growth factor-AA (PDGF-AA) than aortic SMC. Lipid oxidation products accumulate in grafts and may stimulate PDGF production. The effect of oxidized low-density lipoprotein (oxLDL) on PDGF-AA secretion by aortic and graft SMC was compared. SMC isolated from canine thoracic aorta or Dacron thoracoabdominal grafts (n = 10) were incubated with native LDL or oxLDL (0-400 microg/ml) for 72 h. PDGF-AA in the conditioned medium was measured with enzyme-linked immunosorbent assay. OxLDL increased PDGF-AA production by graft SMC from 78 +/- 2 to 256 +/- 16 pg PDGF/microg DNA and aortic SMC from 21 +/- 1 to 40 +/- 2 pg PDGF/microg DNA. Native LDL had no effect. N-acetylcysteine inhibited oxLDL-induced PDGF increase. Both superoxide and H(2)O(2) stimulated PDGF secretion by graft SMC had little effect on aortic SMC. Our results suggest that PDGF production by graft (synthetic) SMC is more sensitive to stimulation by oxidative stress than aortic (contractile) SMC. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress, which stimulates PDGF production by graft SMC. PDGF can induce migration of aortic SMC onto the graft, contributing to the development of intimal hyperplasia.     

Elastogenesis in the wall of the human thoracic aorta

The results of this study dealing with the human thoracic foetal aorta testify that even in the middle of the fifth month of development the internal elastic membrane is not yet completely continuous. Furthermore they show that elastogenesis in the tunica media of the human thoracic aorta does not begin directly below the internal elastic membrane, as it does in the foetal aorta of the laboratory rat, but, as it can be seen in our material, somewhat deeper in the developing tunica media. A thin layer of less differentiated tunica media cells persists for a long time in the vicinity of the internal elastic membrane. In the middle of the fifth month, the fusing elastic membrane segments in the tunica media still consist of very immature elastic tissue with a large proportion of the microfibrillar component. The collagen fibrils in the intercellular spaces in the whole depth of the wall of the developing aorta do not become a part of the elastic membranes. Their bundles merely accompany all the elastic membranes in the wall of the thoracic aorta, including the internal elastic membrane.     

Increased production of prostacyclin (PGI2) by vascular intimal smooth muscle cells from atherosclerotic rabbit aorta

PGI₂ synthesis by aortic strips obtained from thoracic aorta of rabbits fed a high cholesterol diet was examined and compared with that of control rabbits fed a normal diet. In this report, the amounts of PGI₂ produced were shown as 6-keto-PGF_1α per μg of aortic tissue DNA instead of per mg wet weight. We also investigated PGI₂ synthesis by cultured smooth muscle cells (SMC) obtained from atherosclerotic intima.Basal PGI₂ production by aortic strips from atherosclerotic rabbit aorta was significantly augmented compared with that of controls. Arachidonic acid (AA)-induced PGI₂ production by atherosclerotic aorta was also significantly higher than that of controls. PGI₂ producing capacities of intimal and medial layers, separated from atherosclerotic aorta, were examined and the intimal layer was found to elicit a significantly greater PGI₂ production than the medial layer.Furthermore, cultured intimal SMC obtained from atherosclerotic rabbit aorta produced a greater amount of PGI₂ than medial SMC from normal rabbit aorta at various cultured conditions. These results suggest that the possibility of enhanced PGI₂ production by atherosclerotic aorta may well be considered as a defence mechanism of the vessel wall against damaging stimuli.     

Evidence for an age-related dysfunction in the antiproliferative response to transforming growth factor-beta in vascular smooth muscle cells.

Previous studies have indicated that aged animals show an increased intimal hyperplasia after arterial injury. The present studies examined the hypothesis that the increased serum-free proliferation of aged smooth muscle cells (SMC), in vitro, was due to a loss of an antiproliferative signal, such as transforming growth factor-beta 1 (TGF-beta 1). Northern blot analysis of the mRNA derived from old (> 19 mo) or young (3-4 mo) rat aortic SMC indicated that both groups had an equivalent level of the 2.5 kB TGF-beta 1 message. Metabolic labeling with 35S-methionine and immunoprecipitation for TGF-beta 1 confirmed the de novo synthesis of TGF-beta 1 in rat SMC. Old and young SMC supernatants showed equal levels of active or latent (acid-activated) TGF-beta activity. Despite the similarities in the production of TGF-beta 1, old SMC were refractory to inhibition by TGF-beta 1, whereas young SMC were markedly inhibited (80%) by low levels of TGF-beta 1 (IC50 < 5 pg/ml). Binding studies at 4 degrees C indicated that old SMC exhibited reduced binding capacity for 125I-TGF-beta 1. Cross-linking studies confirmed that old SMC showed reduced binding of 125I-TGF-beta 1 to membrane sites corresponding to the high molecular weight type III receptor, as well as the 85-kDa type II and 65-kDa type I receptor. However, at 37 degrees C, old SMC degraded 125I-TGF-beta 1 more rapidly than young SMC. Combined, this data suggests that SMC derived from older animals are capable of normal production of TGF-beta 1 but fail to respond to the autocrine growth inhibitory effects of this agent, thereby leading to enhanced proliferation.     

Focal adhesion kinase (FAK)-dependent regulation of S-phase kinase-associated protein-2 (Skp-2) stability. A novel mechanism regulating smooth muscle cell proliferation

Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.     

A comparison of oxidized LDL-induced collagen secretion by graft and aortic SMCs: role of PDGF

Smooth muscle cells (SMCs) from prosthetic vascular grafts constitutively secrete higher levels of collagen than aortic SMCs. Lipid oxidation products accumulate in grafts, and we postulated that they stimulate SMC production of collagen. The effect of oxidized low-density lipoprotein (oxLDL) on type I collagen secretion by aortic and graft SMCs was compared. SMCs isolated from the canine thoracic aorta or Dacron thoracoabdominal grafts (n = 10) were incubated with native LDL or oxLDL (0-400 microg cholesterol/ml) for 72 h. Type I collagen in the conditioned medium was measured by ELISA. OxLDL increased collagen production by graft SMCs from 4.1 +/- 0.3 to 11.0 +/- 0.4 ng/microg DNA and by aortic SMCs from 2.3 +/- 0.1 to 3.5 +/- 0.2 ng/microg DNA. Native LDL had little effect. LY-83583, a superoxide generator, stimulated a dramatic increase in collagen secretion by graft SMCs and a smaller but significant elevation by aortic SMCs. OxLDL has been shown to increase PDGF production by graft SMCs, and PDGF can stimulate collagen production. Anti-PDGF antibody inhibited the increase in collagen production by graft SMCs that was stimulated by oxLDL, implicating PDGF as one mechanism of oxLDL-induced collagen production. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress that stimulates PDGF production by graft SMCs that in turn stimulates collagen production, contributing to the progression of intimal hyperplasia.     

Integrin-associated protein stimulates alpha2beta1-dependent chemotaxis via Gi-mediated inhibition of adenylate cyclase and extracellular-regulated kinases.

Integrin-associated protein (IAP/CD47) augments the function of alpha2beta1 integrin in smooth muscle cells (SMC), resulting in enhanced chemotaxis toward soluble collagen (Wang, X-Q., and W.A. Frazier. 1998. Mol. Biol. Cell. 9:865). IAP-deficient SMC derived from IAP(-/-) animals did not migrate in response to 4N1K (KRFYVVMWKK), a peptide agonist of IAP derived from the COOH-terminal domain of thrombospondin-1 (TSP1). When normal SMC were preincubated with 4N1K or an anti-alpha2beta1 function-stimulating antibody, cell migration to soluble collagen was significantly enhanced. 4N1K-induced chemotaxis was blocked by treatment of SMC with pertussis toxin indicating that IAP acts through Gi. In agreement with this, 4N1K evoked a rapid decrease in cAMP levels which was intensified in the presence of collagen, and forskolin and 8-Br-cAMP both inhibited SMC migration stimulated via IAP. 4N1K strongly inhibited extracellular regulated kinase (ERK) activation in SMC attaching to collagen and reduced basal ERK activity in suspended SMC. Pertussis toxin treatment of SMC significantly activated ERK, suggesting that an inhibitory input was alleviated. Inhibition of ERK activity by (a) the MAP kinase kinase (MEK) inhibitor, PD98059, (b) antisense oligonucleotide depletion of ERK, and (c) expression of mitogen-activated protein (MAP) kinase phosphatase-1 in SMC all led to increased migration to collagen, 4N1K, or 4N1K plus collagen. Thus, IAP stimulates alpha2beta1 integrin-mediated SMC migration via Gi-mediated inhibition of ERK activity and suppression of cyclic AMP levels. Both of these signaling pathways could directly modulate the state of the integrin as well as impact downstream components of the cell motility apparatus.     

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.     

Engineered alignment in media equivalents: magnetic prealignment and mandrel compaction.

We predicted and measured the evolution of smooth muscle cell (SMC) orientation in media-equivalents (MEs) for four fabrication conditions (F-, M-, F+, M+) under Free or Mandrel compaction (F/M) with and without magnetic prealignment of the collagen fibrils in the circumferential direction (+/-). Mandrel compaction refers to SMC-induced compaction of the ME that is constrained by having a nonadhesive mandrel placed in the ME lumen. Predictions were made using our anisotropic biphasic theory (ABT) for tissue-equivalent mechanics. Successful prediction of trends of the SMC orientation data for all four fabrication cases was obtained: maintenance of the initial isotropic state for F-, loss of initial circumferential alignment for F+, development of circumferential alignment for M-, and enhancement of initial circumferential alignment for M+. These results suggest two mechanisms by which the presence of the mandrel leads to much greater mechanical stiffness in the circumferential direction reported for mandrel compacted MEs relative to free compacted MEs: (1) by inducing an increasing circumferential alignment of the SMC and collagen, and (2) by inducing a large stress on the SMC, resulting in secretion and accumulation of stiffening components.     

Urotensin II Promotes Atherosclerosis in Cholesterol-Fed Rabbits

Urotensin II (UII) is a vasoactive peptide composed of 11 amino acids that has been implicated to contribute to the development of cardiovascular disease. The purpose of this study was to investigate whether UII affects the development of atherosclerosis in cholesterol-fed rabbits. UII was infused for 16 weeks through an osmotic mini-pump into male Japanese White rabbits fed on a high-cholesterol diet. Plasma lipids and body weight were measured every 4 weeks. Aortic atherosclerotic lesions along with cellular components, collagen fibers, matrix metalloproteinase-1 and -9 were examined. Moreover, vulnerability index of atherosclerotic plaques was evaluated. UII infusion significantly increased atherosclerotic lesions within the entire aorta by 21% over the control (P = 0.013). Atherosclerotic lesions were increased by 24% in the aortic arch (P = 0.005), 11% in the thoracic aorta (P = 0.054) and 18% in the abdominal aorta (P = 0.035). These increases occurred without changes in plasma levels of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or body weight. Immunohistochemical staining revealed that macrophages and matrix metalloproteinase-9 were significantly enhanced by 2.2-fold and 1.6-fold in UII group. In vitro studies demonstrated that UII up-regulated the expression of vascular cell adhesion protein-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells, which was inhibited by the UII receptor antagonist urantide. In conclusion, our results showed that UII promotes the development of atherosclerotic lesions and destabilizes atherosclerotic plaques in cholesterol-fed rabbits.     

In vitro study of low density lipoprotein--collagen interaction

Possibility of LDL--collagen complex formation was investigated in vitro by biochemical assay and electron microscopy. Types I and III collagen isolated from bovine thoracic aorta were incubated with human low density lipoproteins (LDL) at physiological ionic strength, pH and temperature. Biochemical quantification showed that 10-20 micrograms LDL (cholesterol) were bound per 100 micrograms collagen, binding of type III being slightly more pronounced (17%) than that of type I (11%). Binding was in inversely proportional to the extent of fibrillation. The increase of ionic strength and pH reduced the binding, indicating the electrostatic nature of the interaction. These observations suggest a possible trapping mechanism of LDL in the extracellular matrix by means of collagen, which may be relevant for the development of the atherosclerotic lesions.     

Increased thromboxane production after whole body irradiation

Irradiation causes an increase in serum and plasma level of thromboxane in rabbits and an increase in thromboxane synthesis in blood platelets stimulated by thrombin. Thromboxane release from thoracic aorta is also increased upon irradiation of animals. H2O2 which stimulates thromboxane release from thoracic control aorta is without effect in the irradiated one.     

Characterization of the vitamin D-dependent Ca2+-binding sites in rat intestinal Golgi-enriched membrane fractions.

Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules.     

A study of aneurysmal lesions of the aorta using magnetic resonance tomography

Altogether 23 patients with aneurysmal aortic lesions of various sites were investigated using MR-tomography. Sagittal and axial projections were used for visualization of the thoracic aorta, frontal and axial ones--for visualization of the abdominal aorta. As compared to radionuclide and x-ray methods of investigation, MR-tomography was characterized by a high informative value in the detection of aneurysmal lesions. Despite a limited number of patients the authors managed to diagnose aortic stratification which could be well visualized in the abdominal aorta.