Thapsigargin-insensitive calcium pools in vascular smooth muscle cells.

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.     

NMDA Receptor Function in the Prefrontal Cortex of a Rat Model for Attention-Deficit Hyperactivity Disorder

The spontaneously hypertensive rat (SHR) is an accepted model for attention-deficit hyperactivity disorder (ADHD) since it displays the major symptoms of ADHD (hyperactivity, impulsivity, and poor performance in tasks that require sustained attention). We have previously shown that glutamate activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors released significantly more norepinephrine from SHR prefrontal cortex slices than control Wistar-Kyoto (WKY) rats. The aim of this study was to determine whether N-methyl-D-aspartate (NMDA) receptor function is disturbed in the prefrontal cortex of SHR. Prefrontal cortex slices were incubated with 45Ca2+ in the presence or absence of 100 microM NMDA for 2 min. Activation of NMDA receptors stimulated significantly less Ca2+ uptake into prefrontal cortex slices of SHR than control WKY (2.8 +/- 0.17 vs. 3.7 +/- 0.38 nmol/mg protein, respectively, P < 0.05). Basal Ca2+ uptake into SHR slices was not significantly different from WKY. These findings are consistent with suggestions that the intracellular concentration of calcium is elevated and therefore the concentration gradient that drives calcium into the cell is decreased in SHR compared to WKY. Impaired NMDA receptor function in the prefrontal cortex of SHR could give rise to impaired cognition and an inability to sustain attention.     

Calcium currents are altered in the vascular muscle cell membrane of spontaneously hypertensive rats

Calcium currents were recorded during whole-cell voltage clamp in cultured azygos venous muscle cells from 1-3-day-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Different holding potentials were used to separate total cell current into its transient (T) and sustained or long-lasting (L) components. In recordings from 30 WKY and 30 SHR vascular cells, total cell calcium current was the same between cells from normotensive (167 +/- 20 pA) and hypertensive (139 +/- 15 pA) rats. However, the relative proportion of T and L calcium currents was different between WKY and SHR cells. In WKY cells, the peak amplitude of the L current was less than that of the T current (42 +/- 30% of total current), whereas in SHR cells, the L current was greater (62 +/- 3% of total current). Calcium currents in vascular muscle cells from SHR were activated and inactivated at more positive potentials than in cells from WKY. This study directly compares transmembrane calcium current in isolated cells from WKY and SHR blood vessels and shows that the proportions of T and L calcium channels activated by depolarization are altered in this genetic model of hypertension.     

Reduced calcium sensitivity of dihydropyridine binding to calcium channels in spontaneously hypertensive rats.

To explore the role of calcium channels in hypertension, dihydropyridine ([3H]PN200-110) binding to heart, brain, and skeletal muscle microsomes of 4-, 8- and 15-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was measured. At a constant Ca2+ ion concentration (pCa 3.0), maximal binding (Bmax) of dihydropyridine binding to heart and brain microsomes was significantly enhanced in 8- and 15-week-old SHR compared with WKY rats (p less than 0.01), whereas this phenomenon was not observed in 4-week-old SHR and WKY rats. Bmax and dissociation constant (Kd) values for skeletal muscle microsomes from SHR showed no difference compared with WKY rats irrespective of age. Dihydropyridine binding to heart microsomes, brain microsomes, and solubilized skeletal muscle microsomes exhibited strong calcium dependence. The Ca2(+)-dependent dihydropyridine binding curves for heart showed a Hill slope, and pK 0.5 values for 15-week-old SHR and WKY rats were 0.70 +/- 0.12 and 4.66 +/- 0.12 versus 0.72 +/- 0.12 and 5.66 +/- 0.08 (n = 4, mean +/- SD), respectively, indicating that 15-week-old SHR require 10-fold higher calcium concentration than WKY rats to promote dihydropyridine binding. The pK 0.5 values of calcium for brain and solubilized skeletal muscle calcium channels in 15-week-old SHR were also significantly lower than in WKY rats. This difference first became apparent in SHR and WKY rats as early as 4 and 8 weeks after birth. These results suggest that enhancement of calcium channel density might occur in the heart and brain of SHR in response to elevated blood pressure and that reduced calcium sensitivity of dihydropyridine binding to calcium channels might be a primary characteristic of this rat strain.     

Cellular calcium regulation in hypertension

In vascular muscle cells, two distinct types of functionally important calcium (Ca2+) channels, called transient (T) and sustained (L), are differentiated by dihydropyridine calcium antagonists (CaA). We studied the ratio of T/L Ca2+ channels in isolated, spontaneously contracting azygous venous cells of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by quantitating Ca2+ currents and intracellular Ca2+ release. While total transmembranous Ca2+ current was not different between the two strains, the proportion of Ca2+ currents carried by L-type channels was enhanced in vascular muscle cells from SHR. We have recently compared subcellular distribution of intracellular free Ca2+ concentration in the same cells, at rest and during stimulation, by quantitation with a digital photon-counting camera. Fura-2 fluorescence intensity showed that Ca2+ release was principally from sarcoplasmic reticulum and that cells from SHR had higher levels of Ca2+ upon calcium channel stimulation, especially at the cell periphery. These findings suggest fundamental differences in SHR and WKY vascular muscle cells implicating the importance of changes in calcium channels, modulation of Ca2+ release, and Ca2+ uptake in SHR hypertension.     

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats

OBJECTIVE: To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. RESULTS: Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. CONCLUSIONS: Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.     

Ca2+ buffering function of the sarcoplasmic reticulum is increased in the carotid artery from spontaneously hypertensive rats.

To clarify whether the Ca2+ uptake function of the sarcoplasmic reticulum (SR) during arterial contraction is altered in hypertension, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit SR Ca2+-ATPase, on the contractile responses to Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of carotid arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of Bay k 8644 (1-300 nM) to the strips caused a concentration-dependent contraction that was larger in SHR than in WKY. The contractile responses to Bay k 8644 were augmented by CPA (10 microM) or thapsigargin (100 nM) in both strains. This augmentation was greater in SHR. Each of CPA and thapsigargin induced a relatively transient contraction, and both of these contractions were larger in SHR than in WKY. The basal 45Ca influx in this artery was larger in SHR than in WKY. The addition of caffeine (1-20 mM) caused a transient contraction that was larger in SHR than in WKY. Our results indicate that 1) the large Ca2+ influx during rest in the SHR carotid artery is strongly buffered by Ca2+ uptake into the superficial SR; and 2) the Ca2+ uptake function of the SR during the contraction with Bay k 8644 was increased in SHR compared with WKY. We conclude that the SHR carotid artery has an increased total capacity of SR for Ca2+ storage as an attempt to compensate for the large Ca2+ influx.     

Differential effects of antihypertensive agents on proliferation of vascular smooth muscle cells from spontaneously hypertensive rats.

We have previously shown that Ca-antagonists and alpha-blockers substantially inhibit the cellular proliferation of cultured rat vascular smooth muscle cells (VSMC). This study explored whether these inhibitory effects on cellular proliferation differ between cultured VSMC from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). SHR VSMC proliferated much faster than WKY VSMC in 10% FCS. Cellular proliferation, determined by both cell number count and 3H-thymidine incorporation, was significantly blunted in the presence of either nifedipine (Nif) or bunazosin (Bun). The magnitude of these inhibitory effects was more pronounced for SHR cells than WKY cells (% reduction of 3H-thymidine uptake with Nif: 62.1 +/- 7.8% for SHR vs 75.3 +/- 10.2% for WKY, n = 6, p less than 0.05, and with Bun: 70.2 +/- 7.8% for SHR vs 82.1 +/- 9.9% for WKY, n = 6, p less than 0.05). In contrast, the intracellular water volume was unaffected by these antihypertensive agents based on equilibrium distribution of 3-O-methyl-D-glucose14C. It is concluded that SHR VSMC grow much faster than WKY VSMC and that this abnormality is innate to the SHR cells. It is also concluded that both Ca-antagonists and alpha-blockers exerted a substantial inhibitory effect on cellular proliferation of the cultured VSMC of either SHR or WKY. Furthermore, the greater inhibition of proliferation in the SHR VSMC suggests that Ca mediated- and/or alpha-receptor mediated processes of cellular proliferation of SHR could differ from that of WKY and that these abnormalities may contribute to the hyperproliferative changes of VSMC in this model.     

Depolarization evoked by acetylcholine in mesenteric arteries of hypertensive rats attenuates endothelium-dependent hyperpolarizing factor

OBJECTIVE: During blockade of endothelium-dependent hyperpolarizing factor (EDHF), acetylcholine evoked larger and faster depolarization in mesenteric arteries of spontaneously hypertensive rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. We studied the mechanism underlying this response and its role in the attenuation of EDHF. METHODS: Electrophysiology, computational modelling and myography were used to study changes in membrane potential and effects on contractility. RESULTS: The large acetylcholine-evoked depolarization in SHR was accompanied by contraction, but this was not seen in WKY rats. The depolarization depended on release of intracellular Ca2+ but was unaffected by nonselective cation channel inhibitors, gadolinium, lanthanum or amiloride. The depolarization was significantly reduced by the Ca2+-dependent Cl- channel inhibitors, niflumic acid or flufenamic acid, or alterations in Cl- gradients using bumetanide (Na/K/Cl transporter inhibitor) or external Cl- replacement with isethionate. These drugs altered the time course of EDHF-evoked hyperpolarizations in SHR, making them indistinguishable from those in WKY rats. EDHF-induced relaxation was less sensitive to acetylcholine in SHR than in WKY rats, but this difference was eliminated following artery pretreatment with bumetanide. Computational modelling in which the SHR fast depolarizing response was selectively modulated mimicked physiologically acquired results obtained in SHR and WKY rats during Cl- -channel blockade. CONCLUSIONS: Acetylcholine evokes a fast depolarization in SHR but not in WKY rats, mediated by the opening of Ca2+-dependent Cl- channels. The depolarization is responsible for a constriction that reduces EDHF-mediated relaxation. Data suggest that Ca2+-dependent Cl- channels may provide a novel therapeutic target for improvement of endothelial dysfunction during hypertension.     

Abnormal platelet and lymphocyte calcium handling in prehypertensive rats

We have reported that the basal and stimulated cytosolic free calcium concentrations [( Ca2+]i) are elevated in platelets isolated from 12-14-week-old spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto (WKY) rats. To determine whether altered cell calcium metabolism precedes the development of overt hypertension, we measured [Ca2+]i under resting and stimulated conditions in blood platelets and thymic lymphocytes isolated from 4-week-old prehypertensive SHR and WKY rats. Blood pressure was similar in both groups (SHR 95 +/- 8 versus WKY rats 92 +/- 7 mm Hg). Basal [Ca2+]i in platelets was higher in SHR than WKY rats (63.4 +/- 3.9 versus 54.8 +/- 3.1 nM, p less than 0.003). Also the [Ca2+]i response to thrombin was greater in SHR than WKY rats in both the presence and absence of extracellular calcium. For lymphocytes, although no difference was detected in basal [Ca2+]i, the concanavalin A-induced peak [Ca2+]i was higher for SHR than WKY rats in both calcium-containing and calcium-free media. These results suggest that agonist-stimulated calcium influx and calcium discharge from intracellular stores are enhanced in both platelets and lymphocytes of 4-week-old SHR. We conclude that abnormalities in calcium metabolism in two different cell types precede the development of overt hypertension in the SHR.     

Eicosanoids and membrane properties in arteries of aged spontaneously hypertensive rats

OBJECTIVE: The arteries of aged spontaneously hypertensive rats (SHR) exhibit spontaneous electrical activity together with membrane depolarization. Vascular eicosanoid production is increased in SHR, which is further accelerated with aging. We tested the hypothesis that eicosanoids are involved in spontaneous electrical activity, membrane depolarization or both in mesenteric arteries of aged SHR. DESIGN AND METHODS: Membrane potentials were recorded with microelectrodes from the mesenteric arteries of aged (24 months and older) SHR, aged Wistar-Kyoto (WKY) rats and adult (6- to 8-month-old) SHR. RESULTS: The membrane potential was less negative in aged SHR (-38.5 +/- 0.9 mV) than in either aged WKY rats or adult SHR (-49.8 +/- 0.5 and -47.2 +/- 0.6 mV, respectively; P < 0.05 for both). Spontaneous electrical activity (5-20 mV, 1-7/min) was present only in arteries of aged SHR. Spontaneous electrical activity was not affected by phentolamine, atropine or tetrodotoxin, but was abolished by indomethacin, a cyclooxygenase inhibitor, and ONO-3708, a thromboxane A2/prostaglandin H2 receptor antagonist. Furthermore, indomethacin and ONO-3708 hyperpolarized the membrane by about 5 mV in aged SHR but not in the other two groups. Spontaneous electrical activity was enhanced by a thromboxane A2 analog and prostaglandin H2, and was abolished by a Ca2+ antagonist, nicardipine, and Ca(2+)-free solution. CONCLUSIONS: These findings suggest that cyclooxygenase-dependent eicosanoids contribute importantly to both spontaneous electrical activity and membrane depolarization, presumably through activation of the thromboxane A2/prostaglandin H2 receptor, in mesenteric arteries of aged SHR, and that spontaneous electrical activity is mediated by a Ca2+ influx through voltage-dependent Ca2+ channels.     

Transforming growth factor-beta1 modulates angiotensin II-induced calcium release in vascular smooth muscle cells from spontaneously hypertensive rats

OBJECTIVES: To investigate the role of transforming growth factor-beta1 (TGF-beta1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Cai2+ release induced by angiotensin II (1 micromol/ l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies. RESULTS: Pretreatment of VSMC for 24 h with TGF-beta1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-beta1 did not modify the response of VSMC from WKY rats to angiotensin II. CONCLUSIONS: These results help our understanding of the interactions between the pathways activated by TGF-beta1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.     

Enhanced spontaneous calcium efflux and decrease of calcium-dependent calcium release from the isolated perfused heart of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to clarify the further details of calcium handling in hypertension. DESIGN: By preserving the physiological environment of cell membrane, whole hearts were used for comparison of calcium flux between spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. METHODS: Hearts from SHR and WKY rats were perfused with Krebs-Henseleit solution under constant flow and the effluent collected. RESULTS: After labelling of the heart with 45Ca2+ (100 mumol/l), 45Ca2+ binding was found to be saturated, and washing with calcium-free perfusion solution showed two exponential curves for calcium dissociation, indicating a fast (alpha-) and slow (beta-) phase. The half-lives of the beta-phase for both 4- and 8-week-old SHR were significantly shorter than those for age-matched WKY. Also in this phase, infusion of non-radioactive Ca2+ caused a transient dose-dependent release of 45Ca2+. A significant reduction in the amount of 45Ca2+ release induced by 2 mmol/l Ca2+ was observed in both 4- and 8-week-old SHR compared with age-matched WKY rats. Infusion of lanthanum, caffeine, ionomycin (calcium ionophore) and treatment of the hearts with ethyleneglycol-bis-(beta-aminoethylether)-N,N,N,',N'-tetraac etic acid did not alter 45Ca2+ release by non-radioactive Ca2+. From these observations, 45Ca2+ is presumably released from the intracellular calcium pool, and not from extracellular binding sites or sarcoplasmic reticulum. CONCLUSIONS: These findings suggest that an abnormal calcium-handling defect (enhanced calcium efflux and reduction of membrane-bound Ca2+) exists under physiological conditions before and after the onset of hypertension, and that this may be a primary characteristic of SHR.     

Calcium channel alterations in genetic hypertension

We proposed earlier that voltage-dependent calcium (Ca2+) current is altered in single azygos venous cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). In this study, the effects of different intracellular concentrations of ethylene glycol-bis-N,N,N',N',-tetraacetic acid (EGTA) on Ca2+ currents were investigated. Vascular muscle cells from SHR and WKY rats were equilibrated with pipette solution containing 0.1 mM or 10 mM EGTA. Increasing the EGTA concentration from 0.1 to 10 mM in SHR vascular cells significantly enhanced the peak amplitude of the longer lasting (L) current from 87 +/- 12 pA to 152 +/- 8 pA, while the transient (T) current amplitude was not significantly different (52 +/- 7 pA and 36 +/- 7 pA, respectively). In WKY rat vascular muscle cells, the amplitudes of the T and L currents were not significantly different with the same comparison of intracellular EGTA concentrations. These observations suggest that relatively low intracellular Ca2+ concentrations can more strongly modulate Ca2+ current through the L channel in SHR than WKY rat vascular muscle cells.     

Cation transport and adenosine triphosphatase activity in rat erythrocytes: a comparison of spontaneously hypertensive rats with the normotensive Brown-Norway strain.

The activity of transport adenosine triphosphatases (ATPases) in saponin-treated erythrocytes as well as the passive membrane permeability for 86Rb+ (K+), 45Ca2+ uptake (in the presence of orthovanadate) and the rate of Na(+)-H+ exchange in intact erythrocytes were studied in spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) and Brown-Norway (BN.lx) rats. Higher Na+,K(+)-ATPase activity, lower Ca(2+)-ATPase activity, increased passive K+ permeability and greater 45Ca2+ uptake were observed in erythrocytes from SHR compared with BN.lx rats. Similar differences in the last two parameters were also disclosed by a comparison of SHR and WKY rats. The rate of Na(+)-H+ exchange in SHR erythrocytes was greater than in WKY rats but equal to that of BN.lx rats. A genetic analysis did not reveal a significant correlation between Na(+)-H+ exchange rate and blood pressure in F2 SHR x WKY hybrids.     

Active calcium and sodium transport by cardiac plasma membranes in the genetically hypertensive rat

Active Na+ and Ca2+ transports by sarcolemmal vesicles from young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were compared. The effects of the calmodulin and the calcium antagonist nifedipine on Ca2+ binding ATP-dependent accumulation of Ca2+ were studied at free Ca2+ concentrations of 2.10(-8)M and 4.10(-7)M. 2.10(-7)M calmodulin stimulated Ca2+ binding to SHR membranes up to a level equivalent to that in WKY, whereas it enhanced active Ca2+ transport more in WKY than in SHR, thus suppressing the difference between the two substrains. At a 2.10(-8)M free Ca2+ concentration low concentrations of nifedipine (10(-7) to 10(-6)M) induced an increases in ATP-dependent Ca2+ transport by SHR vesicles. Inhibition of NA+, K+-adenosine triphosphatase activity by ouabain was also studied. Na+, K+ATPase activity in SHR membranes was double that in membranes from WKY (22.1 +/- 2.8 v.s. 11.3 +/- 1.1. mumole Pi/h/mg protein). These differences, observed on 3 week-old rats, before a significant rise blood pressure, may reflect genetic characteristics of these hypertensive-prone rats.     

Cytoplasmic calcium ion elevating factor(s) in spontaneously hypertensive rat serum

We have attempted to characterize the as yet unspecified circulating factor(s) in spontaneously hypertensive rat (SHR) serum that elevate(s) the cytoplasmic calcium ion concentration in vascular smooth muscle cells (VSMC). We measured the cytoplasmic calcium ion concentration in cultured VSMC before and after incubation with serum from SHR or Wistar-Kyoto rats (WKY). Incubation with serum from overtly hypertensive 8-week-old SHR, but not with serum from 8-week-old WKY, significantly elevated the cytoplasmic calcium ion concentration in VSMC from 8-week-old WKY and 4-week-old SHR in a dose- and an incubation period-dependent manner, although no significant elevation occurred in VSMC from 4-week-old WKY. Neither the heat-immobilized nor the trypsinized 8-week-old SHR serum elevated the cytoplasmic calcium ion concentration. Ultrafiltration procedures indicated that the molecular weight of the factor should be 10,000-30,000 daltons. Incubation with serum from 4-week-old SHR or WKY did not elevate the cytoplasmic calcium ion concentration. These results strongly suggest that: (1) a cytoplasmic calcium ion concentration elevating factor exists in serum from 8-week-old SHR but not from 4-week-old SHR, or 4- or 8-week-old WKY; (2) this factor should be of a protein nature, with a molecular weight of 10,000-30,000 daltons; (3) responsiveness to the factor appears in VSMC at a younger age in SHR than in WKY; (4) the factor elevates the cytoplasmic calcium ion concentration to the level determined a priori not by the strain but by the age of the rats from which the VSMC originate; and (5) the a priori determined level increases with the age of the rats.     

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats

OBJECTIVE : Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS : Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS : ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS : We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.     

Calcium buffering of resting, voltage-dependent Ca2+ influx by sarcoplasmic reticulum in femoral arteries from spontaneously hypertensive rats at prehypertensive stage.

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.     

Bumetanide-sensitive sodium-22 transport in vascular smooth muscle cell of the spontaneously hypertensive rat

The effect of bumetanide, a known probe of Na+, K+ cotransport, on 22Na+ uptake and washout was examined in serially passed cultured vascular smooth muscle cells of spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and Wistar rats. In Ca2+-deficient medium, the drug exerted the greatest effect on 22Na+ washout in vascular smooth muscle cells from SHR and the least effect on cells from WKY. The respective mean values for the apparent bumetanide-sensitive 22Na+ washout rate constants (Ke; X 10(-2)/min) were 7.2, 4.3, and 1.7 for cells from SHR, WKY, and Wistar rats. In both 1 mM Ca2+ and Ca2+-deficient medium, in the presence of 1 mM ouabain, vascular smooth muscle cells from SHR had the highest plateau phase of 22Na+ uptake among the three cell preparations. All cells exhibited higher 22Na+ uptake in Ca2+-deficient medium than in 1 mM Ca2+ medium. Under this condition, bumetanide caused an additional rise in steady state 22Na+ uptake that was most pronounced in cells from SHR (21.3% versus 16.6% for Wistar rats and 4.8% for WKY). This finding indicates that a quantitatively greater inhibition of washout than of the uptake component of the bumetanide-sensitive 22Na+ transport occurs in Ca2+-deficient medium. It is concluded that, in Ca2+-deficient medium, the bumetanide-sensitive 22Na+ washout is higher in vascular smooth muscle cells of SHR than in those of normotensive controls and that this phenomenon reflects a higher Na+ turnover in vascular smooth muscle cell in the hypertensive rat strain.