The elevation of the cytoplasmic calcium ions in vascular smooth muscle cells in SHR —measurement of the free calcium ions in single living cells by lasermicrofluorospectrometry

doi:10.1016/S0006-291X(86)80374-6

Biochemical and Biophysical Research Communications

Volume 141, Issue 1, 26 November 1986, Pages 340-345

https://doi.org/10.1016/S0006-291X(86)80374-6 Get rights and content

Summary

We have developed an accurate and sensitive system for the measurement of cytoplasmic free calcium concentrations ([Ca⁺⁺]_i) of a single cell by using UV-laser and Indo-1. By this method, we made the first successful measurement of [Ca⁺⁺]_i of single living vascular smooth muscle cells. [Ca⁺⁺]_i in spontaneóusly hypertensive rats was elevated and maintained after the 6th passage culture. However, [Ca⁺⁺]_i in Goldblatt hypertensive rats was not elevated. Thus, these results suggest that the maintenance of high [Ca⁺⁺]_i levels of vascular smooth muscle cells in spontaneously hypertensive rats is genetically regulated and that it is one of the mechanisms for hypertension.

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Cited by (113)

Differential growth of craniofacial and tibial bones to sympathetic hyperactivity-related hypertension in rats
2019, Archives of Oral Biology
Citation Excerpt :
Previous studies using SHRs have revealed different results when hypertension developed in the SHR. Sugiyama et al. (1986) have shown that SHRs are born normotensive and develop increased blood pressure from 8 weeks of age, reaching values close to 200 mmHg at 12 weeks of age. Nora et al. (2016) have shown that hypertension develops in SHRs by 4–6 weeks of age, which rises further during the course of aging.
To evaluate the effect of sympathetic nervous system hyperactivity on craniofacial skeletal growth in growing spontaneously hypertensive rats (SHRs).
Craniofacial skeletal growth was compared between male SHR and Wistar―Kyoto rats (WKR) using linear measurements on lateral and transverse cephalometric radiographs at the age of 12 weeks. Tibia length was measured as an index of whole body growth. Body weight　and blood pressure were measured from 3 to 12 weeks of age. Bone microstructure in the mandibular condyle and tibia between the two groups was compared at the age of 12 weeks using microcomputed tomography.
The SHRs had a significantly lower body weight than WKRs from 7 weeks of age, and tibial length was significantly smaller in the SHRs than in the WKR at 12 weeks of age. In all SHRs, blood pressure was significantly higher than in WKRs from 3 to 12 weeks of age. Cephalometric analyses revealed decreased measurements of the neurocranium, viscerocranium, and mandible in SHRs, and mandibular growth was most negatively affected in this group. Lastly, in SHRs, microcomputed tomography analyses revealed decreased bone mineral density and bone volume/tissue volume in the mandibular condyle but not in the tibia.
In growing SHRs, hypertension related to the hyperactivity of the sympathetic nervous system reduced craniofacial skeletal growth more than the growth of the tibia.
Evolving mechanisms of vascular smooth muscle contraction highlight key targets in vascular disease
2018, Biochemical Pharmacology
Citation Excerpt :
This problem was first circumvented by administering aequorin into VSM preparations using a transient membrane permeabilization technique [132]. Thereafter, several fluorescent Ca2+ indicators including quin-2, fura-2, and indo-1 have been developed for measuring [Ca2+]c in many cell types including VSM [133–137]. The nonpolar acetoxymethyl ester of Ca2+ indicators is more lipophilic and diffuses into the cell where it is hydrolyzed by intracellular esterases into the more hydrophilic free acid that does not cross the plasma membrane and is trapped inside the cell.
Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca²⁺ has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ stimulates initial Ca²⁺ release from the sarcoplasmic reticulum, and is buttressed by Ca²⁺ influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c), Ca²⁺ removal mechanisms promote Ca²⁺ extrusion via the plasmalemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchanger, and Ca²⁺ uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca²⁺ handling mechanisms help to create subplasmalemmal Ca²⁺ domains. Threshold increases in [Ca²⁺]_c form a Ca²⁺-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin–myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca²⁺]_c, MLC phosphorylation, and force have suggested additional Ca²⁺ sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca²⁺. PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca²⁺ handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca²⁺]_c, PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.
Greater cytosolic and mitochondrial calcium transients in adrenal medullary slices of hypertensive, compared with normotensive rats
2010, European Journal of Pharmacology
Pronounced differences in the kinetics of single-vesicle catecholamine release from adrenal chromaffin cells stimulated with acetylcholine or high potassium (K⁺) have been recently found between normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Such differences could be explained on the basis of distinct mechanisms of calcium (Ca²⁺) handling by chromaffin cells of NWRs and SHRs. We have explored here this hypothesis in adrenal medullary slices loaded with calcium fluorescent probes to measure the changes in Ca²⁺ concentration in the cytosol ([Ca²⁺]_c), endoplasmic reticulum ([Ca²⁺]_er), and mitochondria ([Ca²⁺]_m). We found the following differences on calcium handling in SHRs, as compared with NWR: (i) higher basal [Ca²⁺]_c and basal [Ca²⁺]_m; (ii) greater [Ca²⁺]_c elevations elicited by acetylcholine and K⁺, with faster activation but slower inactivation; (iii) greater [Ca²⁺]_c elevations elicited by CRT (a mixture of caffeine, ryanodine, and thapsigargin) and by the mitochondrial protonophore FCCP (carbonylcyanide p-(trifluoromethoxy) phenylhydrazone). The higher basal [Ca²⁺]_c and [Ca²⁺]_m suggest an enhanced mitochondrial Ca²⁺ uptake, and the greater [Ca²⁺]_c elevations produced by FCCP indicates a higher mitochondrial Ca²⁺ release into the cytosol. This alteration of intracellular Ca²⁺ movements could explain the greater quantal catecholamine release responses seen in SHRs, as compared with NWRs in previous studies. Furthermore, enhanced mitochondrial Ca²⁺ cycling may be the basis for the dysfunction of mitochondrial bioenergetics, reported to be present in hypertensive states.
Contribution of sarcoplasmic reticulum calcium uptake and L-type calcium channels to altered vascular responsiveness in the aorta of renal hypertensive rats
1999, General Pharmacology
This study examined whether alterations in intracellular or extracellular Ca²⁺ mobilization were related to differences in caffeine and phenylephrine (PHE)-induced contractions between two-kidney, one-clip hypertensive (2K-1C) and normotensive (2K) rat aortas. After depletion and reloading of intracellular Ca²⁺ stores, caffeine and PHE-induced contractions in Ca²⁺-free solution were increased in 2K-1C. Thapsigargin reduced the contraction to caffeine in 2K-1C and 2K with similar sensitivity. PHE-induced contraction in 1.6-mM Ca²⁺ solution was decreased in 2K-1C, and nifedipine was less effective in lowering this response. The responsiveness to extracellular Ca²⁺ was decreased in 2K-1C hypertensive rat aortas. Our results indicate an increased intracellular Ca²⁺ stores that are not related to alteration in Ca²⁺-ATPase function and a lower contribution of L-type channels to the contraction of 2K-1C aortas.
Combined SSCP and heteroduplex analysis of the human plasma membrane Ca2+-ATPase isoform 1 in patients with essential hypertension
1999, Biochemical and Biophysical Research Communications
In recent theories concerning the pathogenesis of essential hypertension, altered calcium homeostasis plays an important role. Increased intracellular Ca²⁺ levels have repeatedly been reported in different cell types of hypertensive subjects. In vascular smooth muscle cells the plasma membrane Ca²⁺-ATPase (PMCA) represents the most important Ca²⁺-ejection system. Modifications of this pump therefore have been assumed to increase contractile tone of small vessels. For this reason, the purpose of this study was to determine if genetic alterations in the hPMCA1 gene might be associated with arterial hypertension. For detection of polymorphisms all 22 PMCA1 exons from 44 patients with essential hypertension (based on rigorous clinical data in addition to a positive family history) and from 40 normotensives without a family history of hypertension were PCR amplified and subsequently subjected to combined single-strand conformation polymorphism (SSCP) and heteroduplex (HTX) analysis. Despite the high sensitivity of almost 100%, differences could not be identified between hypertensives and normotensives within the two groups. These data indicate that at least in this population PMCA1 polymorphisms are presumably not related to common forms of essential hypertension. Furthermore, the high degree of evolutionary conservation of the PMCA1 gene underlines the pivotal role of this ATPase for cell physiology.
Activation of Na+,H+ exchanger produces vasoconstriction of renal resistance vessels
1998, American Journal of Hypertension
To evaluate the influence of the sodium/proton exchanger (Na⁺,H⁺ exchanger) on the constriction of rat resistance vessels and on the iliac artery, the isometric vasoconstrictions of renal resistance vessels and strips from iliac artery derived from Wistar-Kyoto rats were measured using a vessel myograph. The Na⁺,H⁺ exchanger was activated by intracellular acidification using propionic acid. Cytosolic pH (pH_i) and cytosolic free sodium concentration ([Na⁺]_i) in vascular smooth muscle cells were measured using the fluorescent dye technique. The activation of the Na⁺,H⁺ exchanger increased the [Na⁺]_i by 12.4 ± 1.3 mmol/L (n = 8). The activation of the Na⁺,H⁺ exchanger caused a contractile response of the renal resistance vessels (increase of tension, 1.5 ± 0.1 × 10⁻³ N; n = 13) and of the rat iliac artery (increase of tension, 7.5 ± 0.8 × 10⁻³ N; n = 5). The contractile response after activation of the Na⁺,H⁺ exchanger was significantly inhibited in the absence of external sodium or in the presence of amiloride, confirming the involvement of the Na⁺,H⁺ exchanger. The contractile response after activation of the Na⁺,H⁺ exchanger was significantly reduced in the absence of external calcium, after inhibition of calcium channels by nifedipine, and in the presence of an intracellular calcium antagonist 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the activation of the Na⁺,H⁺ exchanger consecutively caused transplasma membrane calcium influx. On the other hand, the inhibition of the Na⁺,Ca²⁺ exchanger by NiCl₂ significantly increased the vasoconstriction of renal resistance vessels after activation of the Na⁺,H⁺ exchanger. The activation of the Na⁺,H⁺ exchanger produces vasoconstriction by an increased cytosolic sodium concentration, inhibition of the Na⁺,Ca²⁺ exchanger, and activation of transplasma membrane calcium influx through potential dependent calcium channels.

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Biochemical and Biophysical Research Communications

Summary

References (8)

Life Science.

J. Biol. Chem.

Laser Science Progress Report of IPCR.

Cell Tissue Res.

Cited by (113)

Differential growth of craniofacial and tibial bones to sympathetic hyperactivity-related hypertension in rats

Evolving mechanisms of vascular smooth muscle contraction highlight key targets in vascular disease

Greater cytosolic and mitochondrial calcium transients in adrenal medullary slices of hypertensive, compared with normotensive rats

Contribution of sarcoplasmic reticulum calcium uptake and L-type calcium channels to altered vascular responsiveness in the aorta of renal hypertensive rats

Combined SSCP and heteroduplex analysis of the human plasma membrane Ca<sup>2+</sup>-ATPase isoform 1 in patients with essential hypertension

Activation of Na<sup>+</sup>,H<sup>+</sup> exchanger produces vasoconstriction of renal resistance vessels