Harm J. Knot

Relaxation of Arterial Smooth Muscle by Calcium Sparks

Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca2+-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure

1 The regulation of intracellular [Ca2+] in the smooth muscle cells in the wall of small pressurized cerebral arteries (100‐200 μm) of rat was studied using simultaneous digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2 Elevation of intravascular pressure (from 10 to 100 mmHg) caused a membrane depolarization from ‐63 ± 1 to ‐36 ± 2 mV, increased arterial wall [Ca2+] from 119 ± 10 to 245 ± 9 nM, and constricted the arteries from 208 ± 10 μm (fully dilated, Ca2+ free) to 116 ± 7 μm or by 45 % (‘myogenic tone’). 3 Pressure‐induced increases in arterial wall [Ca2+] and vasoconstriction were blocked by inhibitors of voltage‐dependent Ca2+ channels (diltiazem and nisoldipine) or to the same extent by removal of external Ca2+. 4 At a steady pressure (i.e. under isobaric conditions at 60 mmHg), the membrane potential was stable at ‐45 ± 1 mV, intracellular [Ca2+] was 190 ± 10 nM, and arteries were constricted by 41 % (to 115 ± 7 μm from 196 ± 8 μm fully dilated). Under this condition of ‐45 ± 5 mV at 60 mmHg, the voltage sensitivity of wall [Ca2+] and diameter were 7.5 nM mV−1 and 7.5 μm mV−1, respectively, resulting in a Ca2+ sensitivity of diameter of 1 μm nM−1. 5 Membrane potential depolarization from ‐58 to ‐23 mV caused pressurized arteries (to 60 mmHg) to constrict over their entire working range, i.e. from maximally dilated to constricted. This depolarization was associated with an elevation of arterial wall [Ca2+] from 124 ± 7 to 347 ± 12 nM. These increases in arterial wall [Ca2+] and vasoconstriction were blocked by L‐type voltage‐dependent Ca2+ channel inhibitors. 6 The relationship between arterial wall [Ca2+] and membrane potential was not significantly different under isobaric (60 mmHg) and non‐isobaric conditions (10‐100 mmHg), suggesting that intravascular pressure regulates arterial wall [Ca2+] through changes in membrane potential. 7 The results are consistent with the idea that intravascular pressure causes membrane potential depolarization, which opens voltage‐dependent Ca2+ channels, acting as ‘voltage sensors’, thus increasing Ca2+ entry and arterial wall [Ca2+], which leads to vasoconstriction.

EXTRACELLULAR K+-INDUCED HYPERPOLARIZATIONS AND DILATATIONS OF RAT CORONARY AND CEREBRAL ARTERIES INVOLVE INWARD RECTIFIER K+ CHANNELS

1. The hypothesis that inward rectifier K(+) channels are involved in the vasodilatation of small coronary and cerebral arteries (100‐200 microm diameter) in response to elevated [K+]o was tested. The diameters and membrane potentials of pressurized arteries from rat were measured using a video‐imaging system and conventional microelectrodes, respectively. 2. Elevation of [K+]o from 6 to 16 mM caused the membrane potential of pressurized (60 mmHg) arteries to hyperpolarize by 12‐14 mV. Extracellular Ba(2+) (Ba2+(o)) blocked K(+)‐induced membrane potential hyperpolarizations at concentrations (IC(50), 6 microM) that block inward rectifier K(+) currents in smooth muscle cells isolated from these arteries. 3. Elevation of [K+]o from 6 to 16 mM caused sustained dilatations of pressurized coronary and cerebral arteries with diameters increasing from 125 to 192 microm and 110 to 180 microm in coronary and cerebral arteries, respectively. Ba2+(o) blocked K(+)‐induced dilatations of pressurized coronary and cerebral arteries (IC50, 3‐8 microM). 4. Elevated [K+]o‐induced vasodilatation was not prevented by blockers of other types of K(+) channels (1 mM 4‐aminopyridine, 1 mM TEA+, and 10 mu M glibenclamide), and blockers of Na(+)‐K(+)‐ATPase. Elevated [K+]o‐induced vasodilatation was unaffected by removal of the endothelium. 5. These findings suggest that K+(o) dilates small rat coronary and cerebral arteries through activation of inward rectifier K(+) channels. Furthermore, these results support the hypothesis that inward rectifier K(+) channels may be involved in metabolic regulation of coronary and cerebral blood flow in response to changes in [K+]o.

Ryanodine receptors regulate arterial diameter and wall [Ca2+] in cerebral arteries of rat via Ca2+-dependent K+ channels

1 The effects of inhibitors of ryanodine‐sensitive calcium release (RyR) channels in the sarcoplasmic reticulum (SR) and Ca2+‐dependent potassium (KCa) channels on the membrane potential, intracellular [Ca2+], and diameters of small pressurized (60 mmHg) cerebral arteries (100‐200 μm) were studied using digital fluorescence video imaging of arterial diameter and wall [Ca2+], combined with microelectrode measurements of arterial membrane potential. 2 Ryanodine (10 μM), an inhibitor of RyR channels, depolarized by 9 mV, increased intracellular [Ca2+] by 46 nM and constricted pressurized (to 60 mmHg) arteries with myogenic tone by 44 μm (≈22 %). Iberiotoxin (100 nM), a blocker of KCa channels, under the same conditions, depolarized the arteries by 10 mV, increased arterial wall calcium by 51 nM, and constricted by 37 μm (≈19 %). The effects of ryanodine and iberiotoxin were not additive and were blocked by inhibitors of voltage‐dependent Ca2+ channels. 3 Caffeine (10 mM), an activator of RyR channels, transiently increased arterial wall [Ca2+] by 136 ± 9 nM in control arteries and by 158 ± 12 nM in the presence of iberiotoxin. Caffeine was relatively ineffective in the presence of ryanodine, increasing [calcium] by 18 ± 5 nM. 4 In the presence of blockers of voltage‐dependent Ca2+ channels (nimodipine, diltiazem), ryanodine and inhibitors of the SR calcium ATPase (thapsigargin, cyclopiazonic acid) were without effect on arterial wall [Ca2+] and diameter. 5 These results suggest that local Ca2+ release originating from RyR channels (Ca2+ sparks) in the SR of arterial smooth muscle regulates myogenic tone in cerebral arteries solely through activation of KCa channels, which regulate membrane potential through tonic hyperpolarization, thus limiting Ca2+ entry through L‐type voltage‐dependent Ca2+ channels. KCa channels therefore act as a negative feedback control element regulating arterial diameter through a reduction in global intracellular free [Ca2+].

Chloride channel blockers inhibit myogenic tone in rat cerebral arteries

1 We have investigated the role of chloride channels in pressure‐induced depolarization and contraction of cerebral artery smooth muscle cells. 2 Two chloride channel blockers, indanyloxyacetic acid (IAA‐94) and 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulphonic acid (DIDS), caused hyperpolarizations (10–15 mV) and dilatations (up to 90 %) of pressurized (80 mmHg), rat posterior cerebral arteries. Niflumic acid, a blocker of calcium‐activated chloride channels, did not affect arterial tone. 3 Dilatations to IAA‐94 and DIDS were unaffected by potassium channel blockers, but were prevented by elevated potassium. IAA‐94 and DIDS had no effect on membrane potential or diameter of arteries at low intravascular pressure, where myogenic tone is absent. Reduction of extracellular chloride (60 mm Cl−) increased the pressure‐induced contractions. Removal of extracellular sodium did not affect the pressure‐induced responses. 4 Our results suggest that intravascular pressure activates DIDS‐ and IAA‐94‐sensitive chloride channels to depolarize arterial smooth muscle, thereby contributing to the myogenic constriction.

Gender differences in coronary artery diameter reflect changes in both endothelial Ca2+ and ecNOS activity.

Elevation of nitric oxide (NO) release from the vascular endothelium may contribute to some of the gender-associated differences in coronary artery function. The mechanisms by which gender affects NO release from the endothelium of coronary arteries are not known. In this study, endothelial function was examined in pressurized coronary arteries from female and male rats. Diameter and endothelial cell intracellular Ca2+ concentration ([Ca2+]i) in intact arteries, as well as enzymatic activity of endothelial constitutive nitric oxide synthase (ecNOS) in arterial lysates, was measured. Elevation of intravascular pressure to 60 mmHg constricted coronary arteries from female animals less than coronary arteries from male animals (18% and 31% constriction, respectively). The increased arterial diameter of coronary arteries from females was associated with elevated endothelial [Ca2+]i (female 174 nM, male 90 nM; P < 0.001). Elevation of Ca2+ activated ecNOS with a similar slope and half-activation constant ( approximately 160 nM) for both female and male coronary arteries. However, at [Ca2+] > 100 nM, ecNOS activity was significantly higher in coronary arteries from female rats compared with their male equivalents (P < 0.01). Maximal activity for ecNOS at saturating Ca2+ (300 nM) was 37% higher in coronary arteries from female animals compared with male animals (P < 0.05). Thus elevated [Ca2+]i in the endothelium of female coronary arteries alone is predicted to increase the production of NO (by nearly 2-fold). This gender difference combined with increased ecNOS activity at a given [Ca2+] in females indicates that tonic NO production should be nearly threefold greater in female coronary arteries compared with male coronary arteries. We conclude that, in the regulation of endothelial Ca2+ and ecNOS, gender differences contribute significantly to the overall decrease in myogenic tone observed in coronary arteries of females.

Increased Ca2+ sensitivity as a key mechanism of PKC-induced constriction in pressurized cerebral arteries

The effects of activating protein kinase C (PKC) with indolactam V (Indo-V) and 1,2-dioctanoyl-sn-glycerol (DOG) on smooth muscle intracellular Ca2+ concentrations ([Ca2+]i) and arterial diameter were determined using ratiometric Ca2+ imaging and video edge detection of pressurized rat posterior cerebral arteries. Elevation of intraluminal pressure from 10 to 60 mmHg resulted in an increase in [Ca2+]i from 74 +/- 5 to 219 +/- 8 nM and myogenic constriction. Application of Indo-V (0.01-3 microM) or DOG (0.1-30 microM) induced constriction and decreased [Ca2+]i to 140 +/- 11 and 127 +/- 12 nM, respectively, at the highest concentrations used. In the presence of Indo-V, the dihydropyridine Ca2+-channel-blocker nisoldipine produced nearly maximum dilation and decreased [Ca2+]i to 97 +/- 7 nM. In alpha-toxin-permeabilized arteries, the constrictor effects of Indo-V and DOG were not observed in the absence of Ca2+. Both PKC activators significantly increased the degree of constriction of permeabilized arteries at different [Ca2+]i. We conclude that 1) Indo-V- or DOG-induced constriction of pressurized arteries requires Ca2+ influx through voltage-dependent Ca2+ channels, and 2) PKC-induced constriction of pressurized rat cerebral arteries is associated with a decrease in [Ca2+]i, suggesting an increase in the Ca2+ sensitivity of the contractile process.

Activation of multiple mitogen-activated protein kinase signal transduction pathways by the endothelin B receptor requires the cytoplasmic tail

Endothelin is a 21-amino acid peptide with remarkably diverse biological properties, including potent vasoconstriction, induction of mitogenesis, and a role in the development of blood vessels. In the present study, stimulation of the endothelin B receptor was found to activate three distinct mitogen-activated protein kinase signal transduction pathways, the extracellular regulated kinase (ERK) 2, c-Jun N-terminal kinase 1 (JNK), and p38 kinase. These mitogen-activated protein kinase isozymes are thought to mediate very different biological outcomes, suggesting that the observed pattern of kinases activation may be important for the diverse biological properties of endothelin. The cytoplasmic tail of the endothelin B receptor was found to be required for activation of all three mitogen-activated protein kinases and stimulation of intracellular calcium levels. An endothelin B receptor truncated at the C-terminal tail was not able to stimulate the mitogen-activated protein kinases or increase cytosolic free calcium. Furthermore, ectopic expression of the cytoplasmic tail attenuated signaling through the wild type receptor. The observed ERK activation appeared to be mediated by heterotrimeric G proteins, since ectopic expression of a transducin α-subunit inhibited endothelin-stimulated ERK activation. The data suggest that the cytosolic tail of the endothelin B receptor is involved in calcium mobilization and mitogen-activated protein kinase activation via a G protein-dependent mechanism.

Potential Cardioprotective Effects of Estrogen Involving Ion Channels, Endothelium, and Coronary Artery Reactivity

The protective effects of estrogen have been studied extensively in the cardiovascular system, where reproductive hormones are known to have major influence on morbidity and mortality. Epidemiological data indicate that women in their reproductive years have a much lower incidence of coronary disease than men of similar age, an advantage that diminishes rapidly with the onset of menopause (1,2). These studies demonstrate a direct correlation between plasma estrogen levels and coronary disease among these populations. A component of the cardioprotective effect of estrogen appears to be related to favorable effects on lipid profiles, which results in less atherosclerotic disease. Estrogen may also have antiproliferative effects by interfering with fibroblast activity that may account for part of the antiatherogenic activity of this hormone (3). Direct or indirect effects of estrogen may also lead to decreased platelet and monocyte adhesion, and a lower likelihood of thrombosis. A number of studies, however, suggest that effects on coronary artery contractility might also account for a substantial portion of the cardioprotective action of estrogen (1,2). Estrogen appears to reduce coronary vasoconstrictor activity, which may increase coronary blood flow, and/ or decrease the likelihood or severity of an ischemic event in the coronary circulation.

INWARD RECTIFIER POTASSIUM CHANNELS IN RESISTANCE ARTERIES

Inward rectifier potassium channels (Kℝ) are present in cardiac muscle, skeletal muscle, and in some vascular smooth muscle cells. The term inward rectification arises from the observation that when the membrane potential of the cell is controlled (for example, by voltage clamp of the cell) then the Kℝ channel will pass larger inward currents (movement of potassium ions from the extracellular solution into the cell), than outward currents (from the cell to the extracellular solution). However, it is important to note that in physiological potassium gradients and at physiological membrane potentials there will be an electrochemical gradient favouring potassium ions to leave the cell, and the inward rectifier will therefore normally pass an outward, hyperpolarizing membrane current. Many other potassium channels are affected by the voltage across the cell membrane, for example both voltage-activated and calcium-activated potassium channels become more active as the membrane potential becomes more positive. The inward rectifier is unusual in becoming less active as the membrane is depolarized, and more active as the membrane potential is hyperpolarized. In fact this property of inward rectification is central to many of the proposed functions of the channel. Thus in skeletal and cardiac muscle, the suggested roles of the Kℝ include generation of the resting membrane potential, preventing potassium loss from the fibers during long duration action potentials, providing a route for potassium uptake into the cell from the transverse tubules, and preventing hyperpolarization of the membrane potential to values more negative than the potassium equilibrium potential by an active electrogenic Na+/K+ ATPase (4). These functions of the Kℝ may also be important in vascular smooth muscle, for example small arteries subjected to physiological transmural pressures depolarize and develop myogenic tone (1,3). The membrane potential of smooth muscle cells in myogenic arteries is around −40 to −50 mV, and the Kℝ will limit the outward movement of potassium ions from the cell under these depolarized conditions. Because K+ must be accumulated into the cell against its electrochemical gradient, mainly by the Na+/K+ ATPase, a process associated with hydrolysis of ATP, minimizing K+ loss during depolarization is of obvious value in preserving the cell’s energy resources.