Vasopressor peptides and depolarization stimulated Ca2+-entry into cultured vascular smooth muscle

45Ca-uptake was measured in monolayers of cultured rat aortic smooth muscle cells. Sufficient extracellular 45Ca could be removed by a 90 second cold La3+ was to reveal stimulation of 45Ca-uptake by high K+-depolarization and the vasopressor peptides angiotensin II and vasopressin. The high K+-stimulated 45Ca-influx was blocked by a dihydropyridine-type Ca2+-antagonist while that stimulated by angiotensin II or vasopressin was not. The 45Ca-influx stimulated by high K+-depolarization was additive to that stimulated by angiotensin II. Vasopressin and angiotensin II stimulated 45Ca-fluxes were not additive. It is concluded that vasopressor peptides stimulate Ca2+-entry through receptor operated Ca2+-channels which are distinct from voltage gated Ca2+-channels.     

Ca2+ coupling in vascular smooth muscle: Mg2+ and buffer effects on contractility and membrane Ca2+ movements

An examination of the literature, over the past two decades, reveals that (1) in studies of different types of vascular smooth muscles, Mg2+ is often either left out of physiological salt solutions or reduced in concentration compared with that in blood; and (2) when excitation--contraction coupling processes have been examined in isolated vascular tissues and cells, a number of artificial (synthetic) amine and organic zwitterion buffers have often been substituted for the naturally occurring bicarbonate and phosphate anions found in the blood and in cells. The influence of extracellular magnesium ions ([Mg2+]0) on tone, contractility, reactivity, and divalent cation movements in vascular smooth muscles, and how they may relate to certain vascular disease states, is reviewed. Data are presented and reviewed which indicate that many of the most commonly used artificial buffers (e.g., Tris, HEPES, MOPS, Bicine, PIPES, imidazole) can exert adverse effects on contractility and reactivity of certain arterial and venous smooth muscles. The data reviewed herein suggest that [Mg2+]0 and membrane Mg are important in the regulation of vascular tone, vascular reactivity, and in control of Ca uptake, content, and distribution in smooth muscle cells. [HCO3-]0 and (or) PO4(2-) anions may be important for normal maintenance of excitability and reactivity and in the control of Ca uptake, content, and distribution in smooth muscle cells.     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D

Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

MCI-154 activates the Ca2+-activated K+ channel of vascular smooth muscle cells

The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca²⁺-activated K⁺ channel (K_Ca channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 μM MCI-154 activated the K_Ca channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100μM MCI-154 to the cytosolic side activated the K_Ca channel directly, suggesting that the Ca₂₊ sensitivity of the K_Ca channel itself is modulated. Though extracellular application of 100 μM amrinone, a phosphodiesterase inhibitor, activated the K_Ca channel in the cell-attached patch configurations, application of 100 μm amrinone to the cytosolic side could not activate the K_Ca channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca²⁺ sensitivity of the K_Ca channel in vascular smooth muscle cells.     

Endothelin evoked Ca2+ transients and oscillations in A10 vascular smooth muscle cells

Endothelin (200 nM) evoked a rapid rise in [Ca2+]i which was then followed by a maintained elevation of [Ca2+]i. The initial transient can be explained by the release of stored Ca2+ whilst the maintained plateau is likely to be an influx of Ca2+ as it was partially inhibited by nifedipine (5 microM) and the remaining component abolished by the removal of extracellular Ca2+. Vasopressin (1 nM) evoked a similar response which also showed a nifedipine insensitive component to it's plateau phase. Endothelin also evoked oscillations in [Ca2+]i; these where characterised by a rapid rising phase followed by a slower decline, with no 'pacemaker' rise in [Ca2+]i preceding the rising phase. The oscillations were inhibited by the addition of 5 microM nifedipine or the removal of extracellular Ca2+ suggesting they are at least in part dependent on voltage gated Ca2+ entry.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.     

Ca2+ movement in smooth muscle cells studied with one- and two-dimensional diffusion models.

Although many of the processes involved in the regulation of Ca2+ in smooth muscle have been studied separately, it is still not well known how they are integrated into an overall regulatory system. To examine this question and to study the time course and spatial distribution of Ca2+ in cells after activation, one- and two-dimensional diffusion models of the cell that included the major processes thought to be involved in Ca regulation were developed. The models included terms describing Ca influx, buffering, plasma membrane extrusion, and release and reuptake by the sarcoplasmic reticulum. When possible these processes were described with known parameters. Simulations with the models indicated that the sarcoplasmic reticulum Ca pump is probably primarily responsible for the removal of cytoplasmic Ca2+ after cell activation. The plasma membrane Ca-ATPase and Na/Ca exchange appeared more likely to be involved in the long term regulation of Ca2+. Pumping processes in general had little influence on the rate of rise of Ca transients. The models also showed that spatial inhomogeneities in Ca2+ probably occur in cells during the spread of the Ca signal following activation and during the subsequent return of Ca2+ to its resting level.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

Contribution of capacitative and non-capacitative Ca2+-entry to M3-receptor-mediated contraction of porcine coronary smooth muscle

We studied the contribution of store-operated or capacitative Ca2+-entry (SOCE or CCE, respectively) through store-operated Ca2+ channels (SOCCs) and the contribution of Ca2+-entry through receptor-operated, non-selective cation channels (ROCCs or NSCCs, respectively), on the M3-receptor-mediated (270 nM Ach) contractile response of porcine coronary smooth muscle strips by means of the respective inhibitors. In the presence of L-VOCC blockade (1 microM verapamil), LOE 908 (inhibition of NSCCs) decreased the contractile response to 75+/-5% (p<0.01, n=6), 2-APB (inhibition of SOCCs) and SK and F 96365 (inhibition of SOCCs and of NSCCs) decreased the response to 45+/-4% (p<0.001, n=10) and to 23+/-2% (p<0.001, n=5), respectively (control: Ach response in the presence of verapamil alone). In the absence of L-VOCC blockade, LOE 908 reduced the Ach-response to 49+/-7% (p<0.001, n=8) and SK and F 96365 to 3+/-2% (p<0.001, n=4) of control, whereas 2-APB transiently increased the response (peak effect: 130+/-11%; p<0.05, n=8). We conclude: (1) the main source of activator Ca2+ during the M3-receptor-mediated contractile response is the Ca2+ influx through L-VOCCs; (2) however, in the presence of L-VOCC blockade, the contractile response is mainly due to Ca2+-entry through SOCCs; (3) NSCCs may be considerably involved in M3-receptor-mediated contraction as they may serve to depolarize the membrane potential and, thus, to open L-VOCCs; (4) in primary tissue of vascular smooth muscle, both, SOCE and Ca2+-entry through NSCCs are activated during M3-receptor stimulation.