Single Vascular Smooth Muscle Cells Research Articles

Modulation of Ca 2+-activated K + channel activity by tyrosine kinase inhibitors in vascular smooth muscle cell

The effects of the tyrosine kinase inhibitors genistein, lavendustin A, and tyrphostin A25 on Ca 2+-activated K + channel activities in freshly isolated single vascular smooth muscle cells from the rat tail artery were studied by patch clamp recording technique. Genistein (5–50 μM) and lavendustin A (10 μM) increased whole-cell Ca 2+-activated K + channel currents. Increase in single channel activities by genistein and lavendustin A was also observed in excised inside-out patches. Diadzein (15 μM), an inactive analogue of genistein, did not alter channel activities. Tyrphostin A25 (10 nM), which had no significant effect on whole-cell currents in concentrations up to 50 μM, increased the open probability of the channels by 841% in inside-out patches. No potentiation of whole-cell and single channel activities by genistein was observed when ATP was omitted from the intracellular solutions. These observations suggest that tyrosine kinase modulates Ca 2+-activated K + channel activities in vascular smooth muscle cells.

Effect of platelet‐derived growth factor on voltage‐operated calcium channels in rabbit isolated ear artery cells

1. Platelet derived growth factor (PDGF), AB and BB isoforms (100 pM) increased calcium channel currents measured by whole cell voltage clamp technique in single vascular smooth muscle cells isolated from rabbit ear arteries. 2. Tyrphostin-23 (100 microM) a selective inhibitor of protein tyrosine kinases, reduced calcium channel currents. Pre-incubation with tyrphostin-23 prevented PDGF-AB induced increase in calcium channel currents. However, in these same cells 10 nM (+)-202791, a dihydropyridine calcium channel agonist, did increase calcium channel currents. 3. Bistyrphostin (10 microM), a selective inhibitor of epidermal growth factor (EGF)-kinase also reduced calcium channel currents and inhibited PDGF-AB-induced increases in calcium channel currents. 4. Genistein (100 microM) a selective inhibitor of tyrosine kinases, structurally unrelated to the tryphostins, also inhibited calcium channel currents and pre-incubation with genistein prevented the PDGF-AB-induced rise in calcium channel currents. 5. These results indicate that PDGF increases calcium channel currents in vascular smooth muscle. This action of PDGF probably involves a tyrosine kinase.

Halothane and isoflurane decrease the open state probability of K+ channels in dog cerebral arterial muscle cells.

Both halothane and isoflurane evoke cerebral vasodilation. One of the potential mechanisms for arterial vasodilation is enhanced K+ efflux resulting from an increased opening frequency of membrane K+ channels. The current study was designed to determine the effects of volatile anesthetics on K+ channel current in single vascular smooth muscle cells isolated from dog cerebral arteries. Patch clamp recording techniques were used to investigate the effects of volatile anesthetics on macroscopic and microscopic K+ channel currents. In the whole-cell patch-clamp mode, in cells dialyzed with pipette solution containing 2.5 mM EGTA and 1.8 mM CaCl2, depolarizing pulses from -60 to +60 mV elicited an outward K+ current that was blocked 65 +/- 5% by 3 mM tetraethylammonium (TEA). Halothane (0.4 and 0.9 mM) depressed the amplitude of this current by 18 +/- 4% and 34 +/- 6%, respectively. When 10 mM EGTA was used in the pipette solution to strongly buffer intracellular free Ca2+, an outward K+ current insensitive to 3 mM TEA was elicited. This K+ current, which was reduced 51 +/- 4% by 1 mM 4-aminopyridine, was also depressed by 17 +/- 5% and 29 +/- 7% with application of 0.4 and 0.9 mM halothane, respectively. In cell-attached patches using 145 mM KCl in the pipette solution and 5.2 mM KCl in the bath, the unitary conductance of the predominant channel type detected was 99 pS. External application of TEA (0.1 to 3 mM) reduced the unitary current amplitude of the 99 pS K+ channel in a concentration-dependent manner. The open state probability of this 99 pS K+ channel was increased by 1 microM Ca2+ ionophore (A23187). These findings indicate that the 99 pS channel measured in cell-attached patches was a TEA-sensitive, Ca(2+)-activated K+ channel. Halothane and isoflurane reversibly decreased the open state probability (NPo), mean open time, and frequency of opening of this 99 pS K+ channel without affecting single channel amplitude or the slope of the current-voltage relationship. Halothane and isoflurane suppress the activity of K+ channels in canine cerebral arterial cells. These results suggest that mechanisms other than K+ channel opening likely mediate volatile anesthetic-induced vasodilation.

The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels.

Hypoxia causes constriction in small pulmonary arteries and dilatation in systemic arteries. Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary blood flow is controlled in the fetus and by which local lung perfusion is matched to ventilation in the adult. HPV reduces the flow of desaturated blood through underventilated areas of lung. Even though many vasoactive substances have been examined as possible mediators of HPV, these appear more likely to be modulators than mediators. Hypoxic contraction has been demonstrated in single pulmonary vascular smooth muscle cells (PVSMC). The ability to sense changes in oxygen tension is observed in PVSMC and type 1 cells of the carotid body. In both cells, hypoxia has been shown to inhibit an outward potassium current, thus causing membrane depolarization and calcium entry through the voltage-dependent calcium channels. In both cells there is evidence to suggest that changes in the redox status of the oxygen-sensitive potassium channel or channels may control current flow, so that the channel is open when oxidized and closed when reduced. The redox status may be determined by the effects of hypoxia on mitochondrial/peroxisomal function or on the activity of an oxidase similar to NAD(P)H oxidase. More studies are needed to precisely define the individual potassium channels responsive to hypoxia and to confirm the gating mechanism. In systemic arteries hypoxia causes an increased current through ATP-dependent potassium channels and vasodilatation, whereas in the pulmonary arteries hypoxia inhibits potassium current and causes vasoconstriction.

Thin filament regulation of force activation is not essential in single vascular smooth muscle cells.

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.

Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ.

The intermittent rise in intracellular Ca2+ concentration ([Ca2+]i oscillation) has been observed in many types of isolated cells, yet it has not been demonstrated whether it plays an essential role during nerve stimulation in situ. We used confocal microscopy to study Ca2+ transients in individual smooth muscle cells in situ within the wall of small arteries stimulated with perivascular sympathetic nerves or noradrenaline. We show here that the sympathetic adrenergic regulation of arterial smooth muscle cells involves the oscillation of [Ca2+]i that propagates within the cell in the form of a wave. Ca2+ release from intracellular stores plays a key role in the oscillation because it is blocked after the store depletion by ryanodine treatment. Ca2+ influx through the plasma membrane sustains the oscillation by replenishing the Ca2+ stores. These results demonstrate the involvement of [Ca2+]i oscillations in the neural regulation of effector cells within the integrated system.

Mesenteric vascular smooth muscle cells from spontaneously hypertensive rats display increased calcium responses to angiotensin II but not to endothelin-1

To determine the differential calcium responses to two vasoconstrictor peptides, angiotensin II (Ang II) and endothelin-1, in vascular smooth muscle cells derived from mesenteric arteries from young and adult normotensive and hypertensive rats. Effects of Ang II and endothelin-1 on cytosolic free calcium concentration in primary cultured unpassaged single vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto (WKY), Wistar and spontaneously hypertensive rats (SHR) aged 3, 9 and 17 weeks were examined microphotometrically using fura-2 methodology. Basal cytosolic free calcium concentration was significantly increased in cells from SHR aged 9 and 17 weeks compared with cells from age-matched WKY and Wistar rats. Ang II and endothelin-1 significantly increased cell cytosolic free calcium in all rat groups at all ages. Responses to low concentrations of Ang II (1 nmol/l) were significantly higher in cells from SHR aged 9 and 17 weeks than in age-matched controls. This was confirmed in cells from rats aged 17 weeks with full concentration-response curves, which also showed that the pD2 for Ang II for WKY rats was significantly different from that of SHR. In cells from SHR at all ages Ang II-stimulated cytosolic free calcium remained persistently high, whereas in cells from WKY and Wistar rats basal levels were reached within 100 s after the maximal response. Low concentrations of endothelin-1 elicited significantly lower cytosolic free calcium responses in cells from SHR aged 17 weeks compared with age-matched controls. The time course of cytosolic free calcium responses to endothelin-1 were similar in the groups. In primary cultured unpassaged mesenteric vascular smooth muscle cells from adult SHR, cytosolic free calcium concentration responses to Ang II are enhanced, whereas responses to low concentrations of endothelin-1 are slightly reduced. The differential effects of these two vasoconstrictor peptides may contribute to their relative roles in modulating vascular smooth muscle cell cytosolic free calcium in SHR.

Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture.

Cultured smooth muscle cells obtained from rat lung periphery (RPC) and proximal pulmonary artery (RSMC) expressed mRNA for serotonin (5-HT) type 2 receptor (5-HT2) and 5-HT transporter (by Northern blot analysis). Functional expression of these genes was evident since both cell types 1) bound 125I-labeled lysergic acid diethylamide (LSD; 5-HT2 receptor antagonist) that was equally effectively displaced by either ketanserin or mianserin; and 2) transported 5-[3H]HT in an imipramine-sensitive manner. Serotonin (10(-9)-10(-5) M) stimulated DNA synthesis (as measured by [3H]thymidine uptake) in RPC and RSMC. The 5-HT-induced increase in DNA synthesis was significantly inhibited in both cell types by the 5-HT2 receptor antagonist, ketanserin (10(-7)-10(-6) M), and by fluoxetine (10(-6) M), a putative 5-HT transport inhibitor. Acute exposure to 5-HT (1-100 microM) caused an abrupt rise in intracellular calcium ([Ca2+]i) in single pulmonary vascular smooth muscle cells as microspectrofluorometrically determined using the calcium-sensitive dye, fura 2. The 5-HT-induced change in [Ca2+]i was completely abolished in the presence of 10(-6) M ketanserin as well as imipramine or fluoxetine (10(-6) M). The calcium transients due to 5-HT persisted in a Na(+)-free condition (in which the transporter activity was completely abolished) and imipramine and fluoxetine (and ketanserin) were effective inhibitors of 5-HT under these conditions. Therefore, the 5-HT2 receptor, but not the transporter, is responsible for initiating the acute effects (e.g., calcium transients) of 5-HT in cultured rat pulmonary vascular smooth muscle cells and fluoxetine (1 microM) may have 5-HT2-receptor antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

O-288 - Noradrenaline-sensitive but caffeine-insensitive single vascular smooth muscle cells.

O-288 - Noradrenaline-sensitive but caffeine-insensitive single vascular smooth muscle cells.

The action of amlodipine on voltage-operated calcium channels in vascular smooth muscle.

1. Amlodipine, a dihydropyridine derivative largely ionized at physiological pH, inhibited calcium channel currents in single vascular smooth muscle cells isolated from rabbit ear artery in a concentration-dependent manner. 2. Amlodipine inhibited the current-voltage relationship for calcium channel currents across the range of test potentials used. However, the effect of amlodipine was more marked on more depolarized test potentials. Amlodipine also shifted the steady-state inactivation curve for calcium channel currents in a hyperpolarized direction. 3. The potency of amlodipine as determined from the steady-state inhibition of calcium channel current induced by the drug was dependent on the holding potential of the cells. Use of a more depolarized holding potential increased the potency of amlodipine. 4. Onset of amlodipine-induced inhibition was relatively rapid at both -60 mV and -40 mV holding potential. The use of a more depolarized holding potential increased the rate of association of amlodipine. No recovery from amlodipine-induced inhibition was seen over a 20 min period following washout of the drug. 5. In addition to voltage-dependence, the action of amlodipine showed use-dependence, in that the effect of amlodipine was more marked when calcium channel currents were evoked frequently. Increasing the frequency of activation of calcium channel currents did not alter the apparent onset rate of amlodipine-induced inhibition, but increased the degree of inhibition achieved by the drug. 6. The electrophysiological properties of amlodipine, particularly its voltage-dependence are probably important determinants of its action in vivo.

Ionic currents in rat pulmonary and mesenteric arterial myocytes in primary culture and subculture

The electrophysiological properties of cultured single vascular smooth muscle (VSM) cells from rat pulmonary (PA) and mesenteric (MA) arteries were studied using the whole cell patch-clamp technique. Cells were studied at 3-7 days as primary cultures, or were replated after 10-20 days and subcultured for 2-5 days. In the standard physiological bath solution (containing 1.8 mM Ca2+), and with 125 mM K+ + 10 mM ethylene glycol-bis(beta-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA)-filled pipettes, both PA and MA primary cultured cells had high input resistances (mean = 2-3 G omega) and resting membrane potentials of about -40 mV. The cells were clamped at a holding potential of -70 mV. Depolarization to -20 mV or more evoked a transient inward current (Iin) that was eliminated in Ca(2+)-free bath solution; this indicates that Iin was carried by Ca2+. Iin was substantially smaller in subcultured cells from both PA and MA. Depolarization also activated three components of outward current (Iout) in primary cultured PA and MA cells: a rapidly inactivating transient component (Irt), a slowly inactivating transient component (Ist), and a steady-state (noninactivating) component (Iss). All three components of Iout were inhibited to varying degrees by 5 mM 4-aminopyridine and were eliminated by replacing intracellular K+ with Cs+, but were only minimally affected by removal of extracellular Ca2+. These results suggest that this Iout was carried by K+ and was voltage gated. Little external Ca(2+)-dependent Iout was observed under these conditions, but a substantial Ca(2+)-dependent component was seen when the EGTA concentration in the pipettes was reduced to 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

The changes in contractile status of single vascular smooth muscle cells and ventricular cells induced by bPTH-(1–34)

Single smooth muscle cells from rat tail artery and ventricular myocytes from neonatal rat were isolated by repeated enzyme digestion. The change in cell area as determined photographically was used as an index of cell contraction. The photographic areas of single smooth muscle cells bathed in normal Tyrode solution were 403±22 (n=13) square micra. Exposure of smooth muscle cells to a modified Tyrode solution containing 60 mM KCl induced cell contraction. This contraction was inhibited by bPTH-(1–34) at a concentration of 1 μM. The inhibitory effect of bPTH-(1–34) was time-dependent with maximum inhibition at 5 min after administration. The photographic areas of ventricular myocytes bathed in the culture medium without fetal calf serum were 516±47 (n=29) square micra. At a concentration o of 1 μM, bPTH-(1–34) produced a time-dependent contraction in ventricular myocytes as shown by the decrease in the photographic cell area (88±2% of the control value at 15 min, n=9, p<0.01). Furthermore, 1 μM nifedipine inhibited the effect of bPTH-(1–34) on the contraction of ventricular myocytes, indicating that bPTH-(1–34) might exert its action via a calcium channel related mechanism. In addition, bPTH-(1–34) increased the contraction frequency of single ventricular cells, which could also be inhibited by nifedipine. The present study suggests that bPTH-(1–34) directly relaxes pre-contracted single vascular smooth muscle cells and contracts single ventricular myocytes.

Neuropeptide Y potentiates calcium-channel currents in single vascular smooth muscle cells.

The effect of neuropeptide Y (NPY) on Ca(2+)-channel currents in isolated vascular smooth muscle cells was studied with the perforated-patch recording technique. Using Ba2+ (10 mM) as the charge carrier, inward currents sensitive to Cd2+ and nifedipine were potentiated by NPY in a concentration-dependent manner. The threshold concentration for the potentiating effect of NPY was 50 nM and reached a maximum at 150 nM. NPY shifted the steady-state activation curve to less positive membrane potentials by about 6 mV so that the potentiating effect was most prominent near the activation threshold of the current. It had no effect on steady-state inactivation of the current. These results suggest that NPY may potentiate vasoconstriction by promoting calcium entry through L-type voltage-dependent Ca(2+)-channels.

Ca 2+ transients recorded in single voltage clamped vascular smooth muscle cells using the fluorescent Ca 2+ indicator indo-1

Ca 2+ transients recorded in single voltage clamped vascular smooth muscle cells using the fluorescent Ca 2+ indicator indo-1

Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i.

The mechanism of alpha-adrenergic agonist-mediated force generation was investigated in single hyperpermeable vascular smooth muscle cells. By use of a previously described method, force was recorded from individual ferret aortic cells made hyperpermeable by exposure to saponin. When the cells were clamped at pCa 7, addition of protein kinase M (PKM), the constitutively active form of protein kinase C (PKC), caused a sustained increase in force, which was reversible upon addition of the PKC pseudosubstrate inhibitor peptide (PSSI) corresponding to residues 19-31 of PKC. Phenylephrine at pCa 7 caused a dose-dependent increase in contractile force of the hyperpermeable cells, which was reversible on addition of phentolamine. The phenylephrine contraction could also be inhibited by the same concentration of PSSI that was effective toward the PKM-induced contraction. The response of the cells to a constant [phenylephrine] in different Ca buffers showed a lack of Ca dependence between pCa 8.6 and 7.0. The addition of PSSI to unstimulated cells caused a small but significant drop in basal tone. Taken together, these results suggest that a fraction of the basal tone, as well as the phenylephrine contraction that occurs in the skinned cells at constant intracellular free Ca2+ concentration, is the result of activation of a Ca-independent isozyme of PKC.

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)- and caffeine-sensitive cellular Ca2+ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca2+ mobilization were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i) using Fura 2 in Ca(2+)-containing or Ca(2+)-free solution. The resting [Ca2+]i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca2+, 10 microM ACh or 128 mM K+ produced a phasic, followed by a tonic, increase in [Ca2+]i but 20 mM caffeine produced only a phasic increase. In Ca(2+)-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca2+]i rapidly decreased to 102 nM within 5 min, and 10 microM ACh or 20 mM caffeine (but not 128 mM K+) transiently increased [Ca2+]i. Ryanodine (50 microM) greatly inhibited the phasic increase in [Ca2+]i induced by 10 microM ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca2+. By contrast, ryanodine (50 microM) enhanced the tonic increase in [Ca2+]i induced by 128 mM K+ and also by 10 microM ACh in Ca(2+)-containing solution. In Ca(2+)-free solution containing 0.5 mM EGTA, ACh (10 microM) failed to increase [Ca2+]i following application of 20 mM caffeine. The level of [Ca2+]i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 microM ACh in Ca(2+)-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone selectively inhibits L‐type calcium channels in single vascular smooth muscle cells of the rat.

1. The active synthetic N-terminal fragment of bovine parathyroid hormone, bPTH-(1-34) at a concentration of 1 microM, decreased the peak amplitude of the long-lasting (L-type) calcium channel current by 37% (n = 14, P less than 0.01) in rat tail artery smooth muscle cells. By contrast, this fragment of parathyroid hormone (PTH) (1 microM) had no effect on the transient (T-type) calcium channel current in the same cell preparation. 2. The inhibitory effect of bPTH-(1-34) on L-channel currents was reversible and could be antagonized by the L-channel agonist, Bay K 8644. In contrast, bPTH-(1-34) inhibited Bay K 8644-induced amplification of L-channel currents. 3. The inhibitory effect of bPTH-(1-34) on L-Channel currents was dose dependent with a threshold concentration of less than 10(-7), and voltage dependent with increased inhibition at more positive holding potentials. However, this effect of bPTH-(1-34) was not dependent on different pulse lengths or interpulse intervals. 4. The kinetics of deactivation of L-channel currents were not changed although the instantaneous amplitude of the L-channel tail current was reduced by bPTH-(1-34). 5. Application of bPTH-(1-34) antagonists (10(-6) M-bPTH-(3-34) and 10(-5) M-bPTH-(7-34] did not result in any significant change in the magnitude of L-channel currents (n = 15 and n = 7, respectively). 6. Pre-incubation of cells with bPTH-(3-34) for more than 15 min abolished the inhibitory effect of bPTH-(1-34) on L-channel currents. 7. The present study provides direct evidence to demonstrate the PTH, an endogenous circulating hormone, is a selective inhibitor of L-channel currents in vascular smooth muscle cells.

ATP‐Gated Channels in Vascular Smooth Muscle Cells

ATP acting through P2x-purinoceptors activates cation channels with some similarities to the activation of channels gated by acetylcholine and glutamate (channels that can also act as fast excitatory transmitters). These experiments clearly demonstrate an ATP-mediated Ca2+ influx through agonist-gated channels and a consequent elevation of [Ca2+]i in these single vascular smooth muscle cells. The combination of the ability to hold these cells under voltage-clamp and to measure [Ca2+]i simultaneously has allowed us to exclude other possible explanations for the rise in [Ca2+]i under these conditions. Thus, although the major cation entering through the channels is Na+, ATP receptor activation will also generate subtle, localized increases in [Ca2+]. These increases might directly activate contractile proteins or, if insufficient to do this, might upregulate other Ca2(+)-dependent enzymes modulating the contractile process and provide an enhanced source of Ca2+ for uptake into internal Ca2+ stores. Further understanding of the physiological role of this conductance pathway may require the development of specific receptor antagonists or channel blockers.

Spatial dynamics of intracellular calcium in agonist-stimulated vascular smooth muscle cells

Vasoconstrictor agonists stimulate smooth muscle contraction by inducing a rise in intracellular free Ca2+. Digital-imaging microscopy of fura-2 fluorescence from single vascular smooth muscle cells cultured from the human internal mammary artery has allowed us to record the subcellular alterations in Ca2+ that occur immediately after stimulation by receptor agonists. The thrombin-induced rise in cytoplasmic free Ca2+ begins in a discrete region typically located close to the end of the cell. Subsequently, this region of elevated Ca2+ expands until Ca2+ is elevated throughout the cell cytoplasm. The rate of spreading in the region of elevated Ca2+ in a linear direction averaged 10.1 microns/s, enabling it to traverse the length of most cells within approximately 5 s, and involved rises in Ca2+ of between 200 and 500 nM. In some cells, the Ca2+ rise began at both ends and collided midway. Similar dynamic changes in the spatial distribution of Ca2+ were recorded in cells stimulated by acetylcholine. The novel observation that vasoconstrictor agonists induce an elevation of Ca2+ in a localized region which subsequently expands throughout the cytoplasm of single smooth muscle cells may provide new insight into the nature of Ca2+ signaling in vascular tissue.

Abnormal calcium handling in vascular smooth muscle cells of spontaneously hypertensive rats

We have developed a system for measuring the dynamic changes in the cytoplasmic free calcium concentration [( Ca2+]i) in single vascular smooth muscle cells (VSMC) that is highly sensitive and does not cause cellular damage. Marked increases in [Ca2+]i in response to stimulation with caffeine and angiotensin II occurred in some VSMC of 4-week-old spontaneously hypertensive rats (SHR), in which the blood pressure and basal [Ca2+]i levels are not yet elevated. In 8-week-old rats, the basal [Ca2+]i level in VSMC was higher in SHR than in Wistar-Kyoto rats. Although the effects of high blood pressure on [Ca2+]i in vivo were expected to disappear during the passage culture, the [Ca2+]i in the fifth-passage cells was similar to that in the primary cells. These results suggest that the maintenance of high [Ca2+]i levels in VSMC of SHR is genetically regulated and is one of the mechanisms of hypertension in this strain, and that abnormal calcium regulation in VSMC of SHR is expressed even before overt hypertension.