Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria

K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.     

Hypertension: cation transport and the physical properties of erythrocytes

In recent years, many reports have appeared describing altered Na+ and K+ transport in erythrocytes of individuals with essential hypertension. Collectively, the interpretation of these results has been unclear. Our studies revealed that the active ouabain-sensitive K+ influx, the furosemide-sensitive K+ influx and the residual passive K+ influx in both human and rat erythrocytes can vary considerably among individual persons or rats and that these measurements alone can not be used to distinguish normotensive from hypertensive individuals. The only consistent cation transport difference observed was an increased Na+ permeability in spontaneously hypertensive rat (SHR) erythrocytes. We have also examined certain physical properties (equilibrium density distribution and sedimentation velocity) of erythrocytes from normotensive Wistar-Kyoto (WKY) and SHR rats, since these characteristics may be altered in response to abnormalities of ion transport. It was found that the erythrocytes from geographically, environmentally, and age-matched littermates of WKY and SHR rats have identical equilibrium density distributions. It was also found that the density distribution of erythrocytes can vary among geographically dispersed colonies of the same strain of rat, and even among successive litters of the same rat colony. However, the sedimentation time required for erythrocytes to reach their equilibrium density was always shorter in the normotensive WKY samples than in the matched SHR. Utilizing a simple centrifugation method, we were able to clearly show that for any population of erythrocytes with the same upper limit of cell density, normotensive WKY cells always sediment at a faster rate than those of the hypertensive SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Brain synaptosomal Ca2+ uptake: comparison of Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats

1. K(+)-stimulated 45Ca2+ uptake by synaptosomes was measured with respect to the strain differences between Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. 45Ca2+ uptake by synaptosomes isolated from cerebral cortex of SD, WKY and SHR was measured at 15, 30, 60, 120 and 240 sec time periods. 3. The sequence of both the magnitude and rate of resting and depolarization-dependent 45Ca2+ uptake was SHR greater than WKY greater than SD. 4. The fastest rates of resting and depolarization-dependent 45Ca2+ uptake occurred in each rat during the first 15 sec and uptake rates dropped off quickly in both resting and depolarization states. 5. At 15 sec, there were significant differences between SHR and WKY, while there were no significant differences between WKY and SD. 6. The results suggest that an important alteration in Ca2+ channel characteristics may occur in SHR brain synaptosomes.     

Na+/H+ exchange in erythrocytes of spontaneously hypertensive rats: a study in F2 SHR x WKY hybrids.

The rate of proton gradient-induced Na+/H+ exchange in the erythrocytes of SHR was increased by 50-60% as compared to WKY animals. No significant correlation between Na+/H+ exchange and blood pressure was revealed in F2 hybrids of SHR and WKY rats. Na+/H+ exchange rate in the erythrocytes of F2 SHR x WKY hybrids was twice as high as in SHR and three times higher than in WKY rats.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

自发性高血压大鼠心脏与红细胞L-Arg转运的改变

研究自发性高血压大鼠 (spontaneouslyhypertensiverats ,SHR)心脏L 精氨酸 /一氧化氮 (L Arg/NO)系统的改变及其与红细胞L Arg转运的关系。检测 12周龄 (W)、16W、captopril治疗 4周后的 16WSHR (SHR C)及同龄Wistar Kyoto (WKY)大鼠心脏的L Arg转运、tNOS活性、NO 2 NO 3 和cGMP含量以及红细胞L Arg转运的改变。结果显示 ,SHR心室肌组织L Arg高亲和转运成分的最大转运速率 (Vmax)及低亲和转运成分的米氏常数 (Km)均明显低于WKY大鼠 ;但高亲和转运成分的Km 值和低亲和转运成分的Vmax则无明显改变 ;SHR C组的改变基本同 12W组。心肌组织tNOS活性的变化无统计学意义。NO 2 NO 3 及cGMP含量则分别较WKY组降低 2 4 6 %、19 8% (P >0 0 5 ,P <0 0 5 ,12W组 ) ,5 2 5 %、6 0 4% (P <0 0 1,P <0 0 1,16W组 )和 14 8%、2 3 % (P >0 0 5 ,P <0 0 5 ,SHR C组 )。tNOS活性、cGMP含量与LVW/BW呈负相关 ,r=0 45 0 7,P =0 0 5 (NOS) ,r=0 6 898,P <0 0 1(cGMP)。红细胞L Arg转运的改变与心脏一致 ,且其Vmax与心肌组织高亲和转运成分的Vmax呈正相关 ,r=0 5 6 0 6 ,P =0 0 1;与LVW /BW呈负相关 ,r=- 0 6 2 31,P <0 0 1。以上结果表明 ,SHR心室肌组织L Arg/NO系统活动被抑制 ,其抑制程度与心肌肥厚     

C-src locus determines increased rate of Na+,K+-cotransport and increased calcium content in erythrocytes of (SHR x WKY) F2 hybrids

26 male F2 hybrids between spontaneously hypertensive (SHR) and normotensive control (WKY) rats (SHRxWKY)F2 were segregated according to their c-src genotype into SS and WW homozygous groups, corresponding to SHR or WKY and WS heterozygous group. Na, K cotransport in erythrocytes in the WW group was equal to that of WKY and differs significantly from that of WS and SS groups (the rate of Na, K cotransport in latter groups was close to that of SHR). Ca content of RBC in WW group was equal to that of WKY, lower than that of WS and SS groups which in turn was significantly lower than in SHR, indicating polygenic control of the trait. Authors conclude that the c-src locus itself or some other loci inherited in conjunction with c-src determines both the increase of Na, K cotransport and calcium content in erythrocytes of SHR.     

Enhanced thromboxane formation by blood but not whole platelets from spontaneously hypertensive rats.

Increased serum levels of immunoreactive thromboxane B2 (iTXB2) were observed in spontaneously hypertensive rats of the Okamoto-Aoki strain (SHR) compared with normotensive Wistar-Kyoto rats (WKY). Serum iTXB2 levels in whole blood allowed to clot at 37 degrees C for 1 hour were significantly greater in SHR than WKY at 8, 16-20, and 38 weeks of age, whereas formation of iTXB2 by thrombin-stimulated whole platelets from 6 16-week-old SHR and 6 age-matched WKY was 399 +/- 44 and 377 +/- 38 ng/10(9) platelets/30 min, respectively. No significant difference in radioconversion of exogenous arachidonic acid to TXB2 was observed in whole platelets from SHR (18.2 +/- 2.5%, n = 4) and WKY (20.1 +/- 3.0%, n = 4) at 16 weeks of age. These results support the proposal that enhanced ability of blood from SHR to generate iTXB2 is independent of the stage of hypertension development. This enhancement probably depended on factors or blood elements other than platelets since no difference in formation was observed on stimulation of whole platelets.     

Factors involved in the susceptibility of spontaneously hypertensive rats to low K+-induced arrhythmias

Disorders of intracellular Ca2+ homeostasis and intercellular coupling are thought to be crucial in the initiation and maintenance of malignant arrhythmias. The aim of this study was to investigate possible arrhythmogenic factors in spontaneously hypertensive rats (SHR) as well as their susceptibility to low K+-related arrhythmias. The experiments were performed on isolated hearts of 13 weeks-old SHR and age-matched Wistar Kyoto rats (WKY). Equilibration of the heart by Langendorff perfusion with oxygenated, 37 degrees C warm, standard Krebs solution at a constant pressure was followed by perfusion with low K+ solution for 60 min, unless sustained ventricular fibrillation occurred earlier. Electrocardiogram and epicardial monophasic action potentials (MAPs) were continuously monitored for incidence of arrhythmias and action potential changes. Myocardial tissue was taken for ultrastructural analysis and immunodetection of the main gap junction protein, connexin-43. The results showed that hypertrophic hearts of SHR exhibited prolongation of MAPs and a decrease in phosphorylation of connexin-43. Moreover, they were more prone to low K+-induced early after-depolarisations and ventricular premature beats as well as to connexin-43 and ultrastructural alterations than WKY rats. Consequently, the incidence of ventricular tachycardia (70% vs. 50%) and both transient (50% vs. 25%) and sustained (60% vs. 25%) ventricular fibrillation was higher in SHR than WKY rats. The results suggest that both prolongation of MAP and connexin-43 alterations are important arrhythmogenic factors facilitating arrhythmias in the setting of Ca2+ disorders due to hypokalaemia.     

Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [(14)C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na(+) the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [(14)C]-L-leucine uptake in SHR PTE cells was approximately 50% that observed in WKY PTE cells (77+/-4 vs 164+/-7 pmol/mg protein). In the absence of extracellular Na(+) the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K(0.5) values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA significantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na(+)-independent [(14)C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [(14)C]-L-leucine was Na(+)-independent in both SHR and WKY PTE cells. The spontaneous [(14)C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [(14)C]-L-leucine efflux in WKY (EC(50) = 9 microM) was greater than in SHR PTE cells (EC(50) = 41 microM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport.     

The effect of Li+ on Na-Ca exchange in cardiac sarcolemmal vesicles

The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+.     

Effect of 1,25(OH)2 vitamin D3 and ionized Ca2+ on 45Ca uptake by primary cultures of aortic myocytes of spontaneously hypertensive and Wistar Kyoto normotensive rats

The effect of several regulators of whole animal Ca2+ homeostasis on 45Ca uptake by primary cultures of aortic myocytes isolated from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats was examined. Exposure of confluent cells to 1.0, 1.25 or 1.50 mM ionized Ca2+ in serum-free medium for seven days resulted in increased 45Ca uptake at the higher concentrations of Ca2+ in cells of the SHR but not the WKY. 1,25 (OH)2 vitamin D3 (1 ng/ml) for 7 days caused enhanced influx in cells from both the SHR and WKY while parathyroid hormone (1-34) (1 ng/ml) was without effect. The data indicate that humoral factors that serve to regulate whole animal Ca2+ homeostasis may also play a role in the regulation of Ca2+ metabolism of the vascular smooth muscle cell.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+ -transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca2+ + Mg2+)-ATPase activity and in Ca2+ efflux at 37 degrees C, depending on the side of the membrane at which the monovalent cations K+ and Na+ were placed. Under the conditions used, (Ca2+ + Mg2+)-ATPase activity and Ca2+ efflux was highest when K+ (35 +/- 0.5 mM (+/- S.E.), mean of four experiments) was at the inside and Na+ (130 mM) at the outside of the ghost membrane.     

Changes in mitochondrial functionality and calcium uptake in hypertensive rats as a function of age

We studied whether mitochondrial functions and Ca2+ metabolism were altered in Wistar Kyoto normotensive (WKY) and spontaneous hypertensive rats (SHR). Ca2+ uptake was decreased in SHR compared to WKY rats. Accumulation of Ca2+ was more efficient in WKY than in SHR rats. mDeltaPsi was lower in SHR compared to WKY rats. Basal complex IV activity was higher in SHR than WKY rats, whereas basal L-citrulline production, an indicator of nitric oxide synthesis, was decreased in SHR and dependent on Ca2+ concentration (p<0.05). Impact of Ca2+ was counteracted by EGTA. These data show an age-dependent decreased mitochondrial functions in brain mitochondria during hypertension.     

Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.     

Age- and sex-dependent stimulation of calcium uptake in duodenal cells by prolactin in vitamin D-deficient rats

Administration of ovine-prolactin (O-PRL) stimulated Ca2+ uptake in isolated duodenal cells prepared from vitamin D-deficient rats. The time course of this effect was biphasic: uptake activity reached a peak in 2.5 hrs followed by a decrease at 5 hrs to original levels. This stimulatory effect of O-PRL was observed in vitamin D-deficient male, but not in female rats. This stimulatory effect was observed in 16- and 26-week old, but not 9 week old, animals. Increase in Ca2+ uptake in duodenal cells was not due to a decrease in intracellular Ca2+ efflux. We measured serum Ca concentration in vitamin D-deficient female rats and found that serum Ca increased in D-deficient female rats between 16 and 52 weeks whereas a minimal increase was observed in D-deficient male rats. Although prolactin was shown to stimulate duodenal Ca2+ uptake, it appears that the source of the increase in levels of serum Ca in D-deficient female rats was not derived from an increase in Ca2+ uptake by prolactin in duodenum. The increase in serum calcium with time may explain why female D-deficient rats survive longer then male.     

Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging

Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.