Intravascular pressure regulates local and global Ca(2+) signaling in cerebral artery smooth muscle cells.

The regulation of intracellular Ca(2+) signals in smooth muscle cells and arterial diameter by intravascular pressure was investigated in rat cerebral arteries (approximately 150 microm) using a laser scanning confocal microscope and the fluorescent Ca(2+) indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca(2+) spark frequency 2.6-fold, Ca(2+) wave frequency 1.9-fold, and global intracellular Ca(2+) concentration ([Ca(2+)](i)) 1.4-fold in smooth muscle cells, and constricted arteries. Ryanodine (10 microM), an inhibitor of ryanodine-sensitive Ca(2+) release channels, or thapsigargin (100 nM), an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, abolished sparks and waves, elevated global [Ca(2+)](i), and constricted pressurized (60 mmHg) arteries. Diltiazem (25 microM), a voltage-dependent Ca(2+) channel (VDCC) blocker, significantly reduced sparks, waves, and global [Ca(2+)](i), and dilated pressurized (60 mmHg) arteries. Steady membrane depolarization elevated Ca(2+) signaling similar to pressure and increased transient Ca(2+)-sensitive K(+) channel current frequency e-fold for approximately 7 mV, and these effects were prevented by VDCC blockers. Data are consistent with the hypothesis that pressure induces a steady membrane depolarization that activates VDCCs, leading to an elevation of spark frequency, wave frequency, and global [Ca(2+)](i). In addition, pressure induces contraction via an elevation of global [Ca(2+)](i), whereas the net effect of sparks and waves, which do not significantly contribute to global [Ca(2+)](i) in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells.

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activates phospholipase C, shifted Ca(2+) signaling from sparks to waves in the smooth muscle cells of rat cerebral arteries. UTP decreased the frequency of Ca(2+) sparks and transient Ca(2+)-activated K(+) (K(Ca)) currents and increased the frequency of Ca(2+) waves. The UTP-induced reduction in Ca(2+) spark frequency did not reflect a decrease in global cytoplasmic Ca(2+), Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCC), or Ca(2+) load of the sarcoplasmic reticulum (SR), since global Ca(2+) was elevated, blocking VDCC did not prevent the effect, and SR Ca(2+) load did not decrease. However, blocking protein kinase C (PKC) with bisindolylmaleimide I did prevent UTP reduction of Ca(2+) sparks and transient K(Ca) currents. UTP decreased the effectiveness of caffeine, which increases the Ca(2+) sensitivity of ryanodine-sensitive Ca(2+) release (RyR) channels, to activate transient K(Ca) currents. This work supports the concept that vasoconstrictors shift Ca(2+) signaling modalities from Ca(2+) sparks to Ca(2+) waves through the concerted actions of PKC on the Ca(2+) sensitivity of RyR channels, which cause Ca(2+) sparks, and of inositol trisphosphate (IP(3)) on IP(3) receptors to generate Ca(2+) waves.

Calcium sparks in smooth muscle.

Local intracellular Ca(2+) transients, termed Ca(2+) sparks, are caused by the coordinated opening of a cluster of ryanodine-sensitive Ca(2+) release channels in the sarcoplasmic reticulum of smooth muscle cells. Ca(2+) sparks are activated by Ca(2+) entry through dihydropyridine-sensitive voltage-dependent Ca(2+) channels, although the precise mechanisms of communication of Ca(2+) entry to Ca(2+) spark activation are not clear in smooth muscle. Ca(2+) sparks act as a positive-feedback element to increase smooth muscle contractility, directly by contributing to the global cytoplasmic Ca(2+) concentration ([Ca(2+)]) and indirectly by increasing Ca(2+) entry through membrane potential depolarization, caused by activation of Ca(2+) spark-activated Cl(-) channels. Ca(2+) sparks also have a profound negative-feedback effect on contractility by decreasing Ca(2+) entry through membrane potential hyperpolarization, caused by activation of large-conductance, Ca(2+)-sensitive K(+) channels. In this review, the roles of Ca(2+) sparks in positive- and negative-feedback regulation of smooth muscle function are explored. We also propose that frequency and amplitude modulation of Ca(2+) sparks by contractile and relaxant agents is an important mechanism to regulate smooth muscle function.

Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells.

1. The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT-PCR, cDNA cloning and expression techniques. 2. RT-PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found. 3. Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n = 3; P < 0.05). A two-fold difference was detected between Kir2.1 mRNA and beta-actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations. 4. Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+. 5. The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half-block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes. 6. Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle.

Ontogeny of local sarcoplasmic reticulum Ca2+ signals in cerebral arteries: Ca2+ sparks as elementary physiological events.

Ca2+ release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum is a key element of excitation-contraction coupling in muscle. In arterial smooth muscle, Ca2+ release through RyRs activates Ca2+-sensitive K+ (KCa) channels to oppose vasoconstriction. Local Ca2+ transients ("Ca2+ sparks"), apparently caused by opening of clustered RyRs, have been observed in smooth and striated muscle. We explored the fundamental issue of whether RyRs generate Ca2+ sparks to regulate arterial smooth muscle tone by examining the function of RyRs during ontogeny of arteries in the brain. In the present study, Ca2+ sparks were measured using the fluorescent Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Diameter and arterial wall [Ca2+] measurements obtained from isolated pressurized arteries were also used in this study to provide functional insights. Neonatal arteries (<1 day postnatal), although still proliferative, have the molecular components for excitation-contraction coupling, including functional voltage-dependent Ca2+ channels, RyRs, and KCa channels and also constrict to elevations in intravascular pressure. Despite having functional RyRs, Ca2+ spark frequency in intact neonatal arteries was approximately 1/100 of adult arteries. In marked contrast to adult arteries, neonatal arteries did not respond to inhibitors of RyRs and KCa channels. These results support the hypothesis that RyRs organize during postnatal development to cause Ca2+ sparks, and RyRs must generate Ca2+ sparks to regulate the function of the intact tissue.

Voltage dependence of Ca2+ sparks in intact cerebral arteries.

Ca2+ sparks have been previously described in isolated smooth muscle cells. Here we present the first measurements of local Ca2+ transients ("Ca2+ sparks") in an intact smooth muscle preparation. Ca2+ sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). Intracellular Ca2+ concentration ([Ca2+]i) was measured in intact cerebral arteries (40-150 micron in diameter) from rats, using the fluorescent Ca2+ indicator fluo 3 and a laser scanning confocal microscope. Membrane potential depolarization by elevation of external K+ from 6 to 30 mM increased Ca2+ spark frequency (4. 3-fold) and amplitude (approximately 2-fold) as well as global arterial wall [Ca2+]i (approximately 1.7-fold). The half time of decay ( approximately 50 ms) was not affected by membrane potential depolarization. Ryanodine (10 microM), which inhibits RyR channels and Ca2+ sparks in isolated cells, and thapsigargin (100 nM), which indirectly inhibits RyR channels by blocking the SR Ca2+-ATPase, completely inhibited Ca2+ sparks in intact cerebral arteries. Diltiazem, an inhibitor of voltage-dependent Ca2+ channels, lowered global [Ca2+]i and Ca2+ spark frequency and amplitude in intact cerebral arteries in a concentration-dependent manner. The frequency of Ca2+ sparks (<1 s-1 . cell-1), even under conditions of steady depolarization, was too low to contribute significant amounts of Ca2+ to global Ca2+ in intact arteries. These results provide direct evidence that Ca2+ sparks exist in quiescent smooth muscle cells in intact arteries and that changes of membrane potential that would simulate physiological changes modulate both Ca2+ spark frequency and amplitude in arterial smooth muscle.

Voltage-dependent K+ currents in smooth muscle cells from mouse gallbladder.

The ionic mechanisms associated with the control of gallbladder contractility are incompletely understood. One type of K+ current, the voltage-dependent K+ (KV) current, is relatively uncharacterized in gallbladder cells and may contribute to muscular excitability. The main focus of this study was therefore to determine the voltage dependence and pharmacological nature of this K+ current in isolated myocytes from mouse gallbladder, using the patch-clamp technique. Currents through Ca(2+)-activated K+ channels were minimized by buffering of intracellular Ca2+ (20 nM free Ca2+) and by inclusion of 1 mM tetraethylammonium (TEA+) in the bathing solution. With 140 mM symmetrical K+, membrane depolarization increased K+ currents, independent of driving force, as assessed by tail current analysis. Half-maximal activation of K+ currents occurred at approximately 1 mV and increased e-fold per 9 mV. Inactivation also increased on depolarization, with a midpoint of -24 mV. Single KV channels were recorded in the cell-attached configuration, exhibiting a single-channel conductance of 4.9 pS. TEA+ at 10 mM reduced KV currents by 36%. At +50 mV, 1 mM and 10 mM 4-aminopyridine inhibited currents by 18% and 35%, respectively, whereas 1 and 10 mM 3,4-diaminopyridine inhibited currents by 11% and 21%, respectively. Quinine inhibited KV currents (at +50 mV, 100 microM and 1 mM quinine inhibited current by 24% and 70%, respectively). In summary, we describe voltage-activated K+ currents from the mouse gallbladder that are likely to contribute to the control of muscular excitability.

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone.

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.

Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries.

Local Ca2+ transients ("Ca2+ sparks") caused by the opening of one or the coordinated opening of a number of tightly clustered ryanodine-sensitive Ca(2+)-release (RyR) channels in the sarcoplasmic reticulum (SR) activate nearby Ca(2+)-dependent K+ (KCa) channels to cause an outward current [referred to as a "spontaneous transient outward current" (STOC)]. These KCa currents cause membrane potential hyperpolarization of arterial myocytes, which would lead to vasodilation through decreasing Ca2+ entry through voltage-dependent Ca2+ channels. Therefore, modulation of Ca2+ spark frequency should be a means to regulation of KCa channel currents and hence membrane potential. We examined the frequency modulation of Ca2+ sparks and STOCs by activation of protein kinase C (PKC). The PKC activators, phorbol 12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 microM), decreased Ca2+ spark frequency by 72% and 60%, respectively, and PMA reduced STOC frequency by 83%. PMA also decreased STOC amplitude by 22%, which could be explained by an observed reduction (29%) in KCa channel open probability in the absence of Ca2+ sparks. The reduction in STOC frequency occurred in the presence of an inorganic blocker (Cd2+) of voltage-dependent Ca2+ channels. The reduction in Ca2+ spark frequency did not result from SR Ca2+ depletion, since caffeine-induced Ca2+ transients did not decrease in the presence of PMA. These results suggest that activators of PKC can modulate the frequency of Ca2+ sparks, through an effect on the RyR channel, which would decrease STOC frequency (i.e., KCa channel activity).

Polymyxin B has multiple blocking actions on the ATP-sensitive potassium channel in insulin-secreting cells.

The action of polymyxin B (0.1 microM) on ATP-sensitive K+ (K+ATP) channels in RINm5F insulin-secreting cells was investigated by patch-clamp techniques. Using inside-out patches, open-cells and outside-out patches, polymyxin B was found to block K+ATP channels by, on average, approximately 90-95% of the initial control level of channel activity. The effects were rapid in onset, sustained and readily reversible. Similar effects were found in patches excised from cells pretreated overnight with 1 microM of the phorbol ester phorbol myristate acetate (PMA). External block of channels was associated with a marked decrease in single-channel current amplitude, whereas these effects were not seen when polymyxin B was added to the inside face of the membrane. In patches bathed with internally applied ATP (0.5 mM) and ADP (0.5 mM), polymyxin B inhibited channels but its actions were not reversible upon removal of the compound. However, when the same protocol was undertaken upon cells pre-treated with PMA, the effects of polymyxin B were readily reversed. Our data suggests that polymyxin B is a novel modulator of K+ATP channels, exhibiting multiple blocking actions that may possibly involve a direct effect upon the channel and indirect effects mediated through the inhibition of endogenous protein kinase(s).

Interaction of diazoxide and cromakalim with ATP-regulated K+ channels in rodent and clonal insulin-secreting cells.

The hyperglycaemia-inducing sulphonamide diazoxide has been previously shown to mediate its effects upon insulin secretion by opening K+ channels and hyperpolarizing the beta-cell membrane. The target site has been characterized as the ATP-regulated K+ (K+ATP) channel protein. In the present study, a detailed investigation of interactions of diazoxide and another K+ channel opener, cromakalim, with K+ATP channels has been performed in individual insulin-secreting cells using patch-clamp techniques. In agreement with previous studies, diazoxide and cromakalim were found to be effective only when ATP was present upon the inside face of the plasma membrane. The ability of both diazoxide and cromakalim to open channels was, however, found to diminish with time following isolation of inside-out patches. Within seconds of forming the recording configuration, the actions of both compounds were potent, and were found to decline steadily as the number of operational channels decreased ('run-down'). In open cells, where the plasma membrane remains partially intact, the rate of run-down was significantly reduced, and effects of channel openers were recorded up to 80 min following cell permeabilization. We also demonstrated that in the absence of ATP, but in the presence of ADP, both diazoxide and cromakalim were able to open K+ATP channels. Interestingly, once the effects of diazoxide and cromakalim on K+ATP channels in the presence of ATP were lost, both compounds opened channels in the presence of ADP. One implication of these data is that the actions of diazoxide and cromakalim involve regulatory proteins associated with the ion channel; this molecule is able to bind ATP, ADP and possibly other cytosolic nucleotides.

Properties of diazoxide and cromakalim-induced activation of potassium channels in cultured rat and RINm5F insulin-secreting cells; effects of GTP.

Experiments have been carried out to examine the effects of GTP on the opening of K+ channels in insulin-secreting cells by diazoxide (0.2 mM) and cromakalim (0.5 mM). Using rat pancreatic beta-cells and RINm5F insulinoma cells, patch-clamp recordings of unitary ATP-sensitive potassium (K+ATP) channel currents were made in either the open cell or outside-out patch recording configurations. Adding diazoxide or cromakalim to either the inside or the outside face of the membrane was found regularly to cause the activation of K+ATP channels in the presence of 0.5 mM ATP. We now demonstrate that in the absence of ATP but in the presence of GTP (0.5-1 mM), both diazoxide and cromakalim activate channels. Effects are rapid in onset, sustained and readily reversible. Both the diazoxide- and cromakalim-induced activation of K+ATP channels were mediated by increases in channel open-state probability, and were not associated with any significant change in either channel amplitude or by an increase in the number of channels in the patch. The actions of both diazoxide and cromakalim were not affected by overnight pretreatment of cells with pertussis toxin, suggesting that PTX-sensitive GTP-binding proteins are not involved in mediating the actions of either compound. These data indicate that diazoxide and cromakalim open K+ATP channels in a manner not solely dependent upon intracellular ATP, but by mechanisms involving other cytosolic nucleotides, including GTP.