Early onset of a decreased intracellular magnesium and phosphate concentration in smooth muscle cell of SHR.

A decrease in total magnesium content is not a direct proof of a decreased magnesium ion concentration. It could reflect a phosphate alteration or an ATP metabolism disorder. Plasma phosphate levels are lower in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto rats (WKYs), and defects in membrane regulation or mitochondrial ATP synthase occur. Only sparse data exist concerning cellular magnesium and phosphate concentrations in hypertensive cells. In aortic smooth muscle cells from 10 SHRs of the Münster strain and 10 age-matched normotensive WKY rats, the intracellular phosphate and magnesium content was measured by electron probe X-ray microanalysis (Camscan CS 24 apparatus, Cambridge, U.K.). The Mg++ content was 0.09 +/- 0.15 g/kg dry weight in SHRs versus 1.15 +/- 0.10 g/kg dry weight in WKY rats (p < 0.01). Vascular smooth muscle phosphate content was 23.6 +/- 0.79 g/kg dry weight in WKY rats versus 15.81 +/- 1.22 g/kg dry weight in SHRs (p < 0.01). In aortic smooth muscle cells of one month old SHRs intracellular magnesium was measured as 1.05 +/- 0.08 versus 1.09 +/- 0.09 g/kg dry weight in WKYs. Intracellular phosphate concentration in one month old SHRs was 18.71 +/- 2.41 versus 21.36 +/- 1.25 g/kg dry weight in WKYs (eight animals in each group). Aortic smooth muscle cells of SHRs are caracterized by markedly lowered intracellular phosphate and magnesium concentrations, resulting in an altered ATP-metabolism, as described earlier. Possibly a membrane defect or a magnesium deficiency or disturbed magnesium channels are responsible for the early onset in the pathogenesis of primary hypertension.

Early-onset increased calcium and decreased magnesium concentrations and an increased calcium/magnesium ratio in SHR versus WKY.

Alterations in the metabolism of calcium and magnesium have been implicated in the pathogenesis of primary hypertension. Calcium influx across the external cellular membrane in smooth muscle cells and cardiomyocytes plays a crucial role in the control of cellular excitation contraction and impulse propagation. Intracellular calcium and magnesium concentrations are controlled by reversible binding to specific calcium binding proteins. The calcium and magnesium flux across the external membrane is regulated by a calcium pump (calcium-magnesium-ATPase), calcium channels and binding to the membrane. In cell membranes and in lymphocytes of essential hypertensives, our group showed increased calcium and decreased magnesium and an increased calcium/magnesium ratio in hypertensive cells. In this context, in aortic smooth muscle cells from 13 spontaneously hypertensive rats (SHR) of the Münster strain (systolic blood pressure 188.4+/-9.8 mmHg) and 13 normotensive rats (NT, systolic blood pressure 118.5+/-7.2 mmHg) aged 9 months, the intracellular calcium and magnesium contents were measured under nearly in vivo conditions by electron-probe microanalysis. Measurements were performed in aortic cryosections 3 microm thick. The calcium content was 124.7+/-4.5* mmol/kg dry weight in SHR versus 110.3+/-4.1 mmol/kg dry weight in NT (Means+/-SD, p < 0.01), the magnesium content was 35.5+/-3.9* in SHR versus 50.1+/-4.9 mmol/kg dry weight in NT /p < 0.01). The calcium/magnesium ratio was significantly increased in SHR versus NT (3.56+/-0.39* versus 2.23+/-0.27, p < 0.01). In hypertensive one month old animals the increase in the calcium/magnesium ratio was not as pronounced as in 9 month old animals. The calcium/magnesium ratio was measured 3.3+/-0.42 in SHR (n = 8) as compared to 2.51+/-0.39 in normotensive animals (n = 8, p < 0.01). Aortic smooth muscle cells from SHR are characterized by markedly elevated intracellular calcium and decreased intracellular magnesium contents compared with normotensive cells. The increased calcium/magnesium ratio in hypertensive cells may be a pathogenetic factor for the development of arteriosclerosis and hypertension.

Importance of decreased intracellular phosphate and magnesium concentrations and reduced ATPase activities in spontaneously hypertensive rats.

A decrease in total magnesium content is not a direct proof of a decreased magnesium ion concentration. It could reflect a phosphate alteration or an ATP metabolism disorder. Plasma phosphate levels are lower in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats, and defects in membrane regulation or mitochondrial ATP synthase occur. Only sparse data exist concerning cellular magnesium and phosphate concentrations in hypertensive cells. In aortic smooth muscle cells from 10 SHRs of the Münster strain and 10 age-matched normotensive WKY rats, the intracellular phosphate and magnesium content was measured by electron probe X-ray microanalysis (Camscan CS 24 apparatus, Cambridge, U.K.). The Mg++ content was 0.90+/-0.15 g/kg dry weight in SHRs versus 1.15+/-0.10 g/kg dry weight in WKY rats (p<0.05). Vascular smooth muscle phosphate content was 23.6+/-0.79 g/kg dry weight in WKY rats versus 15.81+/-1.22 g/kg dryweight in SHRs (p<0.01). In seven animals, erythrocytic ATP content was 180.2+/-102 in SHRs vs. 432+/-72 micromol/L cells in WKY rats (p< 0.01). The Na+/K+-ATPase activity was significantly decreased in hypertensive animals as compared to controls (6.49+/-2.3 vs. 12.64+/-2.9 nmol inorganic phosphate/mg protein/min (p< 0.01)). Aortic smooth muscle cells from SHRs are characterized by markedly lowered cellular phosphate and magnesium concentrations and an altered ATP metabolism, possibly due to a membrane defect or a magnesium deficit in hypertensive cells.

Intracellular Mg++ concentrations in smooth and striated muscle cells in spontaneously hypertensive rats.

Decreased intracellular Mg++ concentrations seem to be involved in the pathogenesis of primary hypertension. Of special interest is the smooth muscle cell with its electrolyte metabolism in primary hypertension, but also heart muscle cells and their Mg++ concentrations are of growing interest. Therefore, in aortic smooth muscle cells and striated heart muscle cells (left ventricle) from 20 spontaneously hypertensive rats (SHR) of the Münster strain and 20 normotensive Wistar-Kyoto rats (WKY), the intracellular Mg++ content was measured. The electron probe x-ray microanalysis technique was used to determine intracellular Mg++ concentrations under nearly in vivo conditions in aortic cryosections 3 microm thick and striated heart muscle cells 4 microm thick (Camscan CS 24 apparatus). Vascular smooth muscle Mg++ content was 36.4 +/- 3.1 mmol/kg dry weight in SHR versus 48.6 +/- 3.7 mmol/kg dry weight in WKY (P < .001). In striated heart muscle cell Mg++ concentrations, there was no significant difference in SHR and WKY (79.9 +/- 5.6 versus 80.3 +/- 5.9 mmol/ kg dry weight). In conclusion, the present study revealed that genetic hypertension in the spontaneously hypertensive rat is accompanied by significantly decreased intracellular Mg++ concentrations in vascular smooth muscle cells. In striated heart muscle cells, Mg++ content was not significantly different in SHR and WKY. Mg++ handling is different in vascular smooth muscle and striated heart muscle cells in WKY and SHR (P < .01).

Na+ and Mg2+ contents in smooth muscle cells in spontaneously hypertensive rats.

Whereas in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content and exchange in vascular smooth muscle cells. Therefore in aortic smooth muscle cells from 10 spontaneously hypertensive rats (SHR) of the Münster strain and 10 normotensive Wistar-Kyoto rats (WKY) aged 8 to 10 months, the intracellular magnesium and sodium content was measured. Electron-probe X-ray microanalysis was used to determine intracellular Mg2+ and Na+ concentrations in aortic cryosections 3 microm thick. The magnesium ion content was 0.90 +/- 0.15 g/kg dry weight in SHR versus 1.15 +/- 0.10 g/kg dry weight in WKY (means +/- SD, P < .05). Vascular smooth muscle sodium ion content was 6.66 +/- 0.39 g/kg dry weight in WKY and 12.61 +/- 0.91 g/kg dry weight in SHR (P < .01). Aortic smooth muscle cells from SHR are characterized by markedly lowered intracellular magnesium ion content and increased sodium ion concentrations in animals 8 to 10 months old, compared with normotensive cells. The results may be due to genetically determined disturbances in transmembrane magnesium and sodium ion transport.

Increased Na+ and decreased Mg2+ intracellular concentrations in vascular smooth muscle cells from spontaneously hypertensive rats.

1. Although in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content and exchange in vascular smooth muscle cells.2. Therefore in aortic smooth muscle cells from 10 spontaneously hypertensive rats (SHR) of the Münster strain and 10 normotensive Wistar-Kyoto rats (WKY) aged 3 and 8-10 months, the intracellular magnesium and sodium content was measured.3.Electron-probe X-ray microanalysis was used to determine intracellular Mg2+ and Na+ concentrations in aortic cryosections 3 micron thick. The Mg2+ content was 47+/-13 mmol/kg dry weight in SHR versus 48+/-19 mmol/kg dry weight in WKY aged 3 months, and 37+/-6 mmol/kg dry weight in SHR versus 47+/-4 mmol/kg dry weight in WKY aged 8-10 months (P<0. 05). Vascular smooth muscle Na+ content was 283+/-59 mmol/kg dry weight in WKY and 402+/-123 mmol/kg dry weight in SHR aged 3 months (P<0.05), and 289+/-17 mmol/kg dry weight in WKY versus 548+/-39 mmol/kg dry weight in SHR aged 8-10 months (P<0.05).4. Aortic smooth muscle cells from SHR are characterized by a markedly lower intracellular Mg2+ content in 8-10-month-old animals and increased Na+ concentrations compared with normotensive cells in 3- and 8-10-month-old rats. The results may be due to genetically determined disturbances in transmembrane Mg2+ and Na+ transport. Cellular magnesium and sodium handling may be disturbed in SHR aortic smooth muscle as it is in hypertensive blood cells. In addition, it is concluded that vascular smooth muscle cell Mg2+-Na+ exchanger can be altered in a subgroup of SHR.

Hard tissue of teeth and their calcium and phosphate content in Ambystoma mexicanum (Urodela: Ambystomatidae).

The wall of the pulp cavity, fracture faces and the demineralized surfaces of teeth from larvae and adults of Ambystoma mexicanum were investigated by scanning electron microscopy (SEM). Calcium and phosphate contents were determined by microanalysis. The apical part of the tooth (crown, tooth apex) contains dentin canals. In the larva, these do not reach the enamel-dentin border but end below this border in front of a denser hard substance, possibly enameloid. The pedicel in the adult and the basal portion of the tooth in the larva (base) are without dentin canals. These parts of the teeth are characterized by longitudinally arranged collagen fibres as visualized on the demineralized surfaces. These observations indicate a congruency in early-larval and adult teeth between base and pedicel as well as apex and crown. This partition is also confirmed by the calcium and phosphate values which were identical in larvae and adults. Highest values are found in enamel and lowest values in the tooth-bearing bone. Calcium and phosphate content show a clear difference between dentin and the basal part of the tooth (pedicel and base). The ring-like dividing zone in the adult tooth is less well mineralized.

Strontium, a tracer to study the transport of calcium in mineralizing tissues by electron probe microanalysis.

In growth plate cartilage the mineralization starts extracellularly in the lower hypertrophic zone. The mineral formed is the calcium phosphate apatite. Enough calcium and phosphate must be available at the mineralization front as well as in regions with proceeding mineralization. There must be a transport of Ca (and phosphate) to these sites. Electron probe X-ray microanalysis is a well established method to analyze element concentrations in small volumes, but it cannot discriminate isotopes. Strontium is similar to Ca in its chemical and biological behaviour and is therefore a suitable tracer to investigate the transport of Ca. Small amounts of Sr (0.1 g per kg body weight) were administered intraperitoneally to young rats. After definite intervals of time ranging from 10 to 120 min, 2-4 rats were killed. On freeze dried cryosections the Sr/Ca ratio of the serum and of the intra- and extracellular space of the growth plate were measured. The Sr/Ca ratio reaches its maximum after about 10 min in the serum and after 20 min in the extracellular space of growth plate cartilage. The intracellular Sr/Ca ratio shows large variations because of the low intracellular Ca and Sr concentration, and is lower than the extracellular ratio for times shorter than 30 min. No significant differences were found between the different cell zones of the unmineralized growth plate cartilage. The results demonstrate that the transport of Ca to the growth plate cartilage is relatively fast and that in growth plate cartilage, Ca is transported extracellularly, not intracellularly.

Electron-probe X-ray microanalysis of sodium ion content in vascular smooth muscle cells from spontaneously hypertensive and normotensive rats.

In aortic smooth muscle cells from 12 spontaneously hypertensive rats (SHR) of the Münster strain and 11 normotensive Wistar-Kyoto rats (WKY), the intracellular Na+ content was measured by electron-probe microanalysis. Measurements were performed in aortic cryosections 3 microns thick; the Na+ content was 12.5 +/- 2.4 g/kg dry weight in SHR versus 6.96 +/- 1.1 g/kg dry weight in WKY (P less than 0.01). Thus, aortic smooth muscle cells from SHR are characterized by a markedly elevated intracellular Na+ content compared with normotensive cells. This may either be due to genetically determined disturbances in transmembrane Na+ transport or to a circulating factor affecting Na+ transport. Cellular Na+ handling may be disturbed in SHR aortic smooth muscle as it is in hypertensive blood cells.

Electron-microscopic and microprobe analyses on the pigmented and unpigmented enamel of Sorex (Insectivora).

Sorex belongs to the Insectivora and has a pigmented tooth enamel due to iron. The pigmented enamel (PE) has a mean Ca/P weight ratio, analyzed by quantitative electronprobe X-ray microanalysis, of about 1.9 (mean molar Ca/P ratio 1.46), and the unpigmented enamel (UE) a Ca/P weight ratio of about 2.0 (mean molar Ca/P ratio 1.59). The PE has a higher iron content (with a value of about 8%) than the UE, as shown by microanalysis of ultrathin sections. Laser microprobe mass analysis (LAMMA) has shown that the carbonate content in the UE is higher than in the PE. In the LAMMA spectrum of the negatively charged ions the carbonate lines could be compared directly with those of negatively charged iron ions. The pigmentation is associated with a low Ca/P ratio but may transfer mechanical strength and acid resistance strength to the PE.

Strontium as a tracer to study the transport of calcium in the epiphyseal growth plate (electronprobe microanalysis).

At the mineralization front of the epiphyseal growth plate large quantities of calcium (Ca) are necessary to form the mineral (a Ca-phosphate). It is an unsolved problem, whether Ca is transported through the cells of the growth plate cartilage or extracellularly. Electronprobe microanalysis (EPMA) allows the quantitative, morphologically correlated analysis of elements. EPMA can discriminate only different elements. To investigate the transport of Ca, Strontium (Sr) is a very good tracer, as it resembles Ca in many biological reactions. Our results demonstrate that the transport of Sr from the blood into the growth plate and through the growth plate needs only one or a few minutes. The measured intracellular Sr and Ca concentrations are much lower than the extracellular ones, while the intracellular Sr/Ca ratio is not or only a little bit lower than the extracellular one. It must be concluded, that significant amounts of Ca are neither transported through nor accumulated in the cells of the growth plate cartilage. The main transport is an extracellular diffusion. Using Sr as a tracer for Ca new results on the behaviour of Ca could be received.

Myocardial intra- and extracellular calcium changes following cardioplegic cardiac arrest.

To study the behavior of myocardial cellular calcium during the course of myocardial ischemia following cardiac arrest induced by the chemical cardioplegia, LK352, transmural biopsies were made from the apex of the left ventricle of nine patients undergoing electric cardiac surgery. The biopsies were made before the onset of extracorporeal circulation, at the end of ischemia, and after the discontinuation of cardiopulmonary bypass following reperfusion and cardiac resuscitation. Ultrastructural studies focused on the pattern of distribution in the interstitial space of a certain combination of elements forming microcrystals, together with a quantitative intracellular calcium assay, based on electron probe X-ray microanalysis, presented the following results: In the course of ischemia, a highly significant depletion of intracellular calcium was associated with the pooling of calcium in the interstitial space. An intracellular calcium assay following coronary reperfusion with systemic blood demonstrated intracellular calcium replenishment in association with the depletion of the calcium which had accumulated in the interstitial space during ischemia. The intracellular concentration of calcium after reperfusion approximated the control value. Based on our results, we consider the intracellular calcium depletion a mechanism which keeps the heart arrested in the diastole.

Does correlation exist between mineralization in collagen-rich hard tissues and that in enamel?

Quantitative electron microscopy has shown that strands of crystallites with a dotlike substructure are formed and arranged in relation to the collagen structure. Many such parallel-arranged crystallites unite rapidly to form ribbons or long platelets which penetrate the microfibrils, producing the positive biomechanical quality. We assume that, in enamel, strands of crystallites with dotlike substructure may also exist in the earliest stage. These substructures unite with neighboring ones to form larger entities. Several needlelike (or ribbonlike) crystallites will form large crystallites, healing most of the lattice defects.

Mineralization of human aortas with coarctation: quantitative electron probe microanalysis.

Aortas with coarctation (isthmus stenosis) are obviously an ideal model to investigate pressure dependent changes of the aorta, as one can compare the proximal region (high pressure) with the distal region (low pressure). 7 aortas of patients aged from 2 months to 54 years were investigated. The concentrations of Na, Mg, P, S, C1, K, and Ca were determined by electronprobe microanalysis. Ca and P are constituent parts of the developing mineral and are mainly discussed. The Ca/P ratio (by weight) is about 0.3 in the unmineralized and about 2 in the mineralized tissue. Mineralization is demonstrated by a parallel increase of Ca, P, and the Ca/P ratio. Enrichments of Ca and P are found above all in a subintimal band. Usually they occur proximally and distally, but are much more pronounced proximally. Even in the aorta of a 2 month old infant enrichments were found proximally, but not distally. Thus mineral deposits occur very early in regions of hypertension. The differences between the proximal and distal region may demonstrate the influence of blood pressure on vascular mineralization. The mineralization starts in small compartments, which increase in size and number in the process of mineralization. Mineralized regions could often be localized by cathodoluminescence. But to demonstrate and localize the initial mineral deposits and to quantify element contents, electronprobe microanalysis became indispensable.

Morphological studies on the epiphyseal growth plate combined with biochemical and X-ray microprobe analyses.

The epiphyseal growth plate of the domestic pig was investigated topologically combining biochemical methods with electron microprobe microanalyses both correlated to histological controls. A lateral resolution of about 50 micrometer was reached. Highest nuclease activity was found in the lower columnar cell zone, while alkaline phosphatase showed maximal activity in the hypertrophic area, connected with maximal values for extractable, organically bound phosphorus, and extractable Ca and Mg. Acid phosphatase activity reached maximal values in the zone of the lower primary spongiosa, while the extractable Pi had maximal values at the end of the zone of bone remodelling. Microprobe analyses have shown that the extracellular Ca content (per dry mass) remained relatively constant at 0.7% (about 58 mM/kg wet weight for 66% tissue fluid) in all zones of the plate increasing to 1% in the vicinity of the first foci of mineralization. The intracellular P content (per dry mass) was about 4.5 %, the extracellular 0.1-0.2% (about 10-20 mM/kg wet weight) increasing also to about 1% in the vicinity of the first foci of mineralization. Thus the Ca X P product was much higher than the ion-product of 2 mM2 which is necessary for an in vitro mineralization of connective tissue. The extracellular S content (per dry mass) as a probable indicator of sulfated proteoglycans was relatively constant at about 3.5% in the different zones but decreased to about 0.3% in the fully mineralized regions. This indicates a loss of sulfur containing substances with mineralization which is not so high since the concentrations per dry mass must be normalized to a unit volume of equal density of mass.

[Electron probe x-ray microanalysis of human aorta with isthmus stenosis]. / Menschliche Aorten mit Isthmusstenose: Elementbestimmungen mit der Elektronenstrahlmikroanalyse.

Aortas with isthmus stenosis (coarctation) can be regarded as a model for those changes of the vessel wall, that depend on blood pressure, as one can compare the proximal region (high pressure) with the distal one (low pressure). The Na, Mg, P, S, Cl, K, and Ca content of the vessel walls of 5 patients (age: 2 months to 34 years) were determined by electron probe microanalysis. We have used cryostat sections and measured the element concentrations from the lumen to the outer media. In this paper we discuss especially the measurements of Ca and P, as the mineral being deposited in the vessel wall, e.g. in arteriosclerosis, contains Ca and P. Up to 18 years Ca and P enrichments are found only in a subintimal band parallel to the lumen. The proximal region of the aorta of a 34 years old patient, however, demonstrated Ca and P enrichments in the whole media with a high peak in the subintimal band. The enrichments are proximally always higher than distally. Only in the distal region of the 2 months old infant no Ca or P enrichments were found, but they appeared proximally. Thus high blood pressure causes an enrichment of Ca and P in a subintimal band very rapidly and promotes the mineralization of the vessel wall. The quantitative electron probe microanalysis demonstrated these pathological alterations.

Coarctation of the aorta. A risk factor in children for the development of arteriosclerosis.

Pressure recordings in 120 patients, aged 0-35 years, undergoing operation for coarctation of the aorta, show a slow but steady rise in both systolic and diastolic peak pressures, the highest peak registered being in patients aged 16-18 years. The finding of significant morphological changes in the aorta in this age group is the rule and these arteriosclerotic changes become more severe as the patients get older. In long-standing coarctation, the degree of mineralisation proximally is up to 10 times higher than distally. The microscopic and ultrastructural changes in the proximal segment are essentially arteriosclerotic. In conclusion, the role of coarctation of the aorta in the pathogenesis of arteriosclerosis is essentially that of hypertension, a most important risk factor of arteriosclerosis. Since arteriosclerosis also develops in the region of low blood pressure, other factors are operative. We suppose that the reduced pulse and volume pressures, which in turn diminish the "windkessel" function of the aorta may effect a change in the blood flow patterns to favour the development of arteriosclerosis.