thapsigargin and iberiotoxin

The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels.

Topics: AMP-Activated Protein Kinases; Animals; Calcium Signaling; Calcium-Binding Proteins; Cells, Cultured; Cricetinae; Enzyme Activation; Indoles; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Membrane Potentials; Mesocricetus; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Peptides; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Vascular Resistance; Vasodilation; Vasomotor System

The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca(2+)](i)).. The authors used primary LC cells obtained from normal human donor eyes. Confocal microscopy was used to investigate the effect of hypotonic cell membrane stretch on cell volume changes. Whole-cell patch-clamp and calcium imaging techniques were used to investigate the effect of hypotonicity on ion channel(s) activity and [Ca(2+)](i) changes, respectively. RT-PCR was used to examine for the maxi-K(+) signature in LC cells.. In this study, LC cells showed significant volume changes in response to hypotonic cell swelling. The authors characterized a large conductance K(+) channel (maxi-K(+)) in LC cells and demonstrated its increased activity during cell membrane hypotonic stretch. RT-PCR revealed the presence of maxi-K(+) signature in LC cells. The authors showed the [Ca(2+)](i) and maxi-K(+) channels to be dependent on extracellular Ca(2+) and inhibited by gadolinium, which blocks stretch-activated channels (SACs). Pretreatment with thapsigargin, which blocks the release of Ca(2+) from endoplasmic reticulum stores, showed no significant difference in [Ca(2+)](i) concentration on hypotonic swelling.. The results show that hypotonic stress of human LC cells activates SAC and Ca(2+)-dependent maxi-K(+) channels and that the increase in [Ca(2+)](i) during cell swelling was predominantly from extracellular sources (or intracellular stores other than the endoplasmic reticulum). These findings improve the understanding of how LC cells respond to cell membrane stretch. Further experiments in this area may reveal future targets for novel therapeutic intervention in the management of glaucoma.

Topics: Biomarkers; Calcium; Cell Culture Techniques; Cell Membrane; Cell Size; DNA Primers; Gadolinium; Humans; Hypotonic Solutions; Ion Channels; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channels; Male; Microscopy, Confocal; Optic Disk; Patch-Clamp Techniques; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scorpion Venoms; Stress, Physiological; Thapsigargin

Bradykinin (BK) is an inflammatory mediator that can cause bronchoconstriction. In this study, we investigated the membrane currents induced by BK in cultured human airway smooth muscle (ASM) cells. Depolarization of the cells induced outward currents, which were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner with an IC50 of 0.33 microM. The currents were increased by elevating intracellular free Ca2+ concentration, suggesting they are calcium-activated potassium channels [I(K(Ca))]. Preexposure to inhibitor of I(K(Ca)) of large conductance (BKCa), iberiotoxin, and small conductance (SKCa), apamin, inhibited the increase of outward current induced by BK. The relative contribution of BKCa was greatest in early passage cells. Both nickel and SKF-96365 (10 microM) inhibited the increase of the I(K(Ca)) induced by BK; however, the l-type Ca2+ channel blocker, nifedipine, had no effect. Activation of the BK-induced current was inhibited by heparin, indicating dependence on intact inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca2+ stores. BK also increased inositol phosphate accumulation and induced a transient Ca2+-activated chloride current (CACC) and a sustained nonselective cation current (I(CAT)). In summary, BK activates BKCa, SKCa, CACC, and I(CAT) via IP3-sensitive stores in human ASM.

Topics: Apamin; Bradykinin; Bronchi; Calcium Signaling; Cells, Cultured; Humans; Inositol 1,4,5-Trisphosphate Receptors; Large-Conductance Calcium-Activated Potassium Channels; Muscle, Smooth; Nifedipine; Peptides; Potassium Channels, Calcium-Activated; Ryanodine Receptor Calcium Release Channel; Small-Conductance Calcium-Activated Potassium Channels; Tetraethylammonium; Thapsigargin

Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca(2+) influx is triggered by NE, the Ca(2+) entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca(2+) influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca(2+) response after repetitive stimulation. Ca(2+) entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca(2+) entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca(2+) entry. Moreover, NE failed to induce further Ca(2+) entry after the development of Ca(2+) entry induced by thapsigargin (Tg), suggesting that the pathway of Ca(2+) entry induced by NE might be identical to that of Tg. Interestingly, Ca(2+) entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca(2+)-activated K(+) (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca(2+) entry coupled to BK channels and that NE-induced Ca(2+) entry is important in the regulation of AANAT.

Topics: Adrenergic alpha-Agonists; Animals; Arylalkylamine N-Acetyltransferase; Calcium; Enzyme Inhibitors; Female; Fluorescent Dyes; Fura-2; Male; Melatonin; Membrane Potentials; Norepinephrine; Peptides; Pineal Gland; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Thapsigargin

Although rat aorta smooth muscle cells in culture constitutively express bradykinin B1 receptors, the normotensive rat aorta does not respond to the bradykinin B1 receptor agonist des-Arg9-bradykinin, whereas vessels from the spontaneously hypertensive rat (SHR) respond to bradykinin B1 receptor agonists with cell membrane hyperpolarization and relaxation. Bacterial lipopolysaccharide also is inactive on the normotensive rat but hyperpolarizes the SHR aorta. To determine whether this could be due to the increased intracellular Ca2+ concentration ([Ca2+]i) in the SHR, we raised [Ca2+]i in normotensive rats by treatment with thapsigargin. In the thapsigargin-treated aorta, both lipopolysaccharide and des-Arg9-bradykinin induced hyperpolarization, which was reversed by the Ca2+-dependent K+ channel inhibitor iberiotoxin and by the bradykinin B1 receptor antagonists Lys-[Leu8]-des-Arg9-bradykinin and [Leu8]-des-Arg9-bradykinin. Thus the bradykinin B1 receptor, as well as lipopolysaccharide, needs activated Ca2+-dependent K+ channels for functional expression. The two bradykinin B1 receptor inhibitors, however, have effects on Ca2+-dependent K+ channels which are not mediated by bradykinin B1 receptors.

Topics: Animals; Aorta; Bradykinin; Bradykinin B1 Receptor Antagonists; Calcium; Calcium-Transporting ATPases; In Vitro Techniques; Kallidin; Ligands; Lipopolysaccharides; Male; Membrane Potentials; Myocytes, Smooth Muscle; Peptides; Potassium Channels, Calcium-Activated; Rats; Rats, Wistar; Receptor, Bradykinin B1; Thapsigargin

The aim of this study was to characterize the effects of hypotonicity on the activity of large-conductance Ca(2+)-activated K+ (BK(Ca)) channels in human retinal pigment epithelial (RPE R-50) cells. Effects of hypotonicity on ion currents were investigated with the aid of the patch-clamp technique. A regulatory volume decrease in response to a hypotonic solution (200 mOsm/L) was observed that could be blunted by paxilline. In whole-cell current recordings, a hypotonic solution (200 mOsm/L) reversibly increased the amplitude of K+ outward currents (I(K)). The increase of I(K) could be reversed by iberiotoxin (200 nM), paxilline (1 microM), or tetrandrine (5 microM), but not by glibenclamide (10 microM), disulphonic acid (DIDS) (100 microM), or dequalinium dichloride (10 microM). In RPE R-50 cells pretreated with thapsigargin, aristolochic acid, or pertussis toxin, the increased amplitude of I(K) in response to hypotonicity was unaltered. In cell-attached patches, an increase in BK(Ca)-channel activity was observed during hypotonicity-induced cell swelling. The enhanced channel activity elicited under this condition was mainly mediated by an increase in the number of long-lived openings. These findings support the evidence for the coupling of volume swelling to the functional activity of BK(Ca) channels.

Topics: Alkaloids; Aristolochic Acids; Benzylisoquinolines; Calcium; Calcium Channel Blockers; Cell Membrane Permeability; Cell Size; Cells, Cultured; Humans; Hypotonic Solutions; Indoles; Lactones; Membrane Potentials; Ophthalmic Solutions; Osmolar Concentration; Patch-Clamp Techniques; Peptides; Pertussis Toxin; Pigment Epithelium of Eye; Potassium; Potassium Channel Blockers; Potassium Channels, Calcium-Activated; Signal Transduction; Thapsigargin

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2 sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2 on the cytosolic Ca2+ concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]i in control but not PHTN PA SMC. In control PA SMC, [Ca2+]i increased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+ channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCa blockade had no effect, whereas Kv blockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ ATPase activity caused a greater increase in [Ca2+]i in controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]i in PHTN compared with control PA SMC. KCa channel mRNA is decreased and Kv channel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2 sensing, alters intracellular Ca2+ homeostasis, and changes the predominant ion channel that determines basal [Ca2+]i from KCa to Kv.

Topics: Animals; Blood Proteins; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Cytoplasm; Enzyme Inhibitors; Female; Fetal Diseases; Fetus; Hypertension, Pulmonary; Hypoxia; Muscle, Smooth, Vascular; Oxygen; Peptides; Potassium; Potassium Channels; Pregnancy; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sheep; Thapsigargin

We sought to elucidate the regulation of gallbladder smooth muscle (GBSM) excitability by localized Ca(2+) release events (sparks) and large-conductance Ca(2+)-dependent (BK) channels by determining whether sparks exist in GBSM and, if so, whether they activate BK channels. Sparks were identified in isolated GBSM loaded with fluo 4. Each spark was associated with a transient outward current, suggesting communication of ryanodine receptor (RyR) channels with BK channels. This was confirmed by the inhibition of outward currents with iberiotoxin (100 nM), thapsigargin (200 nM), and ryanodine (10 microM). In current clamp mode, the transient BK currents were associated with brief membrane hyperpolarizations (10.9 +/- 1.3 mV). Because transient BK currents could dampen GBSM excitability, we tested whether CCK attenuates these events. CCK (10 nM) reduced the amplitude and frequency of transient BK currents, and subsequent caffeine application restored transient BK current activity. These results support the concept that RyRs and BK channels contribute to the regulation of GBSM excitability and that CCK can act in part by inhibiting this pathway.

Topics: Aniline Compounds; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Cholecystokinin; Enzyme Inhibitors; Fluorescent Dyes; Gallbladder; Guinea Pigs; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle, Smooth; Nifedipine; Patch-Clamp Techniques; Peptides; Phosphodiesterase Inhibitors; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Thapsigargin; Xanthenes

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.. 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.. 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.. 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.

Topics: Animals; Arteries; Calcium; Carotid Arteries; Charybdotoxin; Hypertension; In Vitro Techniques; Mesenteric Arteries; Peptides; Potassium Channels, Calcium-Activated; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reference Values; Rest; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin; Vasoconstriction

Large-conductance Ca(2+)-dependent K(+) (BK(Ca)) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BK(Ca) currents and intracellular Ca(2+) revealed that BK(Ca) currents are activated by Ca(2+) release events (Ca(2+) sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca(2+) sparks and BK(Ca) currents and to determine the voltage dependence of the coupling of RyRs (Ca(2+) sparks) to BK(Ca) channels in UBSM. Ca(2+) sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca(2+) sparks caused BK(Ca) currents at all voltages tested, consistent with the BK(Ca) channels sensing approximately 10 microM Ca(2+). Membrane potential depolarization from -50 to -20 mV increased Ca(2+) spark and BK(Ca) current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca(2+) spark amplitude increasing by only 30% and BK(Ca) current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BK(Ca) current was quantitatively explained by the known voltage dependence of the Ca(2+) sensitivity of BK(Ca) channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca(2+) sensitivity of BK(Ca) channels, profoundly alters the coupling strength of Ca(2+) sparks to BK(Ca) channels.

Topics: Animals; Calcium; Electrophysiology; Enzyme Inhibitors; Guinea Pigs; Membrane Potentials; Muscle, Smooth; Peptides; Potassium Channel Blockers; Potassium Channels; Ryanodine Receptor Calcium Release Channel; Thapsigargin; Urinary Bladder

1. This study investigates the mechanism of CGRP-induced relaxation in intramural coronary arteries by determining the effect of CGRP on cytosolic Ca(2+) concentration ([Ca(2+)](i)) using FURA-2 technique. 2. CGRP concentration-dependently (10 pM - 100 nM) decreased the [Ca(2+)](i) and tension of coronary arteries precontracted with either U46619 or BAY K 8644, and also of resting coronary arteries in PSS. In 36 mM K(+)-depolarized arteries, CGRP reduced only the tension without affecting the [Ca(2+)](i). 3. In 300 nM U46619- precontracted arteries, pretreatment with 10 microM thapsigargin significantly (P<0.05) attenuated the CGRP-induced reduction in the tension (but not [Ca(2+)](i)). 4. In 300 nM U46619-precontracted arteries, pretreatment with either 100 nM charybdotoxin or 100 nM iberiotoxin or 10 nM felodipine significantly (P<0.05) attenuated the CGRP-induced reduction in both [Ca(2+)](i) and tension. In contrast, 1 microM glibenclamide did not affect the CGRP-induced responses in these coronary arteries. 5. In resting coronary arteries, only pretreatment with the combination of 1 microM glibenclamide and 100 nM charybdotoxin attenuated the CGRP-induced decrease in the [Ca(2+)](i) and tension, suggesting a different mechanism of action for CGRP in resting coronary arteries. 6. We conclude that CGRP relaxes precontracted rat coronary arteries via three mechanisms: (1) a decrease in [Ca(2+)](i) by inhibiting the Ca(2+) influx through membrane hyperpolarization mediated partly by activation of the large conductance Ca(2+)-activated potassium channels, (2) a decrease in [Ca(2+)](i) presumably by sequestrating cytosolic Ca(2+) into thapsigargin-sensitive Ca(2+) storage sites and (3) a decrease in the Ca(2+)-sensitivity of the contractile apparatus. In resting coronary arteries, however, there seems to be an interplay between different types of K(+) channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Analysis of Variance; Animals; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Agonists; Charybdotoxin; Coronary Vessels; Drug Interactions; Enzyme Inhibitors; Felodipine; Fluorescent Dyes; Fura-2; Glyburide; Hypoglycemic Agents; In Vitro Techniques; Male; Osmolar Concentration; Peptides; Potassium; Potassium Channels; Rats; Rats, Sprague-Dawley; Rest; Thapsigargin; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

We have previously identified a human vascular smooth muscle clone that can reversibly convert between proliferative and contractile phenotypes. Here we compared receptor-channel coupling in these cells using fura-2 to monitor [Ca(2+)](i) and patch-clamp to record currents. Histamine elevated [Ca(2+)](i) in all cells and caused contraction of cells exhibiting the contractile phenotype. The rise of [Ca(2+)](i) persisted in Ca(2+)-free solution and was abolished by thapsigargin, indicating involvement of stores. Whole cell electrophysiological recording revealed that histamine evoked transient outward K(+) current, indicating functional receptor-channel coupling. The time-course and amplitude of the histamine-activated current were similar in cells of the proliferative and contractile phenotypes. Moreover, a large conductance K(+) channel was recorded in cell-attached patches and was activated by histamine as well as the Ca(2+) ionophore A-23187, identifying it as the large conductance Ca(2+)-dependent K(+) channel. This K(+) channel showed similar characteristics and activation in both proliferative and contractile phenotypes, indicating that expression was independent of phenotype. In contrast, histamine also elicited an inward Cl(-) current in some contractile cells, suggesting differential regulation of this current depending on phenotype. These studies demonstrate the usefulness of this human vascular cell clone for studying functional plasticity of smooth muscle, while avoiding complications arising from extended times in culture.

Topics: Calcimycin; Calcium; Cell Division; Cells, Cultured; Chlorides; Enzyme Inhibitors; Histamine; Humans; Ionophores; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Muscle Contraction; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Peptides; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Tetraethylammonium; Thapsigargin; Vasoconstriction

Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly by Ca(2+) release and removal to and from the cytoplasm (Somlyo and Somlyo. J Cardiovasc Pharmacol 8, Suppl 8: S42-S47, 1986) by buffering Ca(2+) influx and contributing to Ca(2+) extrusion (Mueller and van Breemen. Nature 281: 682-683, 1979) and indirectly by releasing Ca(2+) near Ca(2+)-activated K(+) channels (K(Ca)) to hyperpolarize the plasma membrane (Bolton and Imaizumi. Cell Calcium 20: 141-152, 1996 and Nelson et al. Science 270: 633-637, 1995). In the rabbit basilar artery, relative contributions of direct effects and those mediated through activation of K(Ca) were evaluated by measuring force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to the SR-depleting agents thapsigargin and ryanodine and the large conductance K(Ca) (BK(Ca)) blockers iberiotoxin (IbTX) and tetraethylammonium ion (TEA). A large contraction was observed in response to K(Ca) blockade with either 3 mM TEA or 100 nM IbTX and also after addition of 10 microM ryanodine or 2 microM thapsigargin. When K(Ca) was blocked first with TEA or IbTX, subsequent addition of thapsigargin or ryanodine also increased force. Measurements of fura 2 fluorescence showed parallel increases in [Ca(2+)](i) in response to sequential blockade of sarco(endo)plasmic reticulum Ca(2+)-ATPase and K(Ca) regardless of the order of application. It appears that a significant fraction of K(Ca) remains activated in the absence of SR function and that SR contributes to relaxation after blockade of K(Ca). We found that depletion of SR before stimulating Ca(2+) influx through voltage-gated Ca(2+) channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms.

Topics: Animals; Basilar Artery; Buffers; Calcium; Calcium Channels; Enzyme Inhibitors; In Vitro Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Tetraethylammonium; Thapsigargin; Vasoconstriction

The effects of tetrandrine, a blocker of voltage-dependent Ca(2+) channels, on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In whole-cell configuration, tetrandrine (0.5-50 microM) reversibly decreased the amplitude of K(+) outward currents. The IC(50) value of tetrandrine-induced decrease in outward current was 5 microM. The K(+) outward current in response to depolarizing voltage pulses was also inhibited by iberiotoxin (200 nM), yet not by glibenclamide (10 microM) or apamin (200 nM). The reduced amplitude of outward current by tetrandrine can be reversed by the further addition of Evans' blue (30 microM) or niflumic acid (30 microM). Thus, the tetrandrine-sensitive component of outward current is believed to be Ca(2+)-activated K(+) current. Pretreatment with thapsigargin (1 microM) or sodium nitroprusside (10 microM) for 5 h did not prevent tetrandrine-mediated inhibition of outward current. In outside-out configuration, bath application of tetrandrine (5 microM) did not change the single-channel conductance but significantly reduced the opening probability of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. The tetrandrine-mediated decrease in the channel activity was independent on internal Ca(2+) concentration. Tetrandrine (5 microM) can also shift the activation curve of BK(Ca) channels to more positive potentials by approximately 20 mV. The change in the kinetic behavior of BK(Ca) channels caused by tetrandrine is due to a decrease in mean open time and an increase in mean closed time. The present study provides substantial evidence that tetrandrine is capable of suppressing the activity of BK(Ca) channels in endothelial cells. The direct inhibition of these channels by tetrandrine should contribute to its effect on the functional activities of endothelial cells.

Topics: Alkaloids; Apamin; Benzylisoquinolines; Calcium; Calcium Channel Blockers; Cell Line; Dose-Response Relationship, Drug; Drug Interactions; Endothelium, Vascular; Evans Blue; Glyburide; Humans; Membrane Potentials; Niflumic Acid; Nitroprusside; Peptides; Potassium Channels; Thapsigargin; Umbilical Veins

This study investigated nitroglygerin (NTG) relaxations in isolated dog coronary artery in comparison with other vascular preparations. Under maximal PNU-46619 precontraction, the coronary artery was significantly more sensitive to NTG than mesenteric artery, mesenteric vein and saphenous vein. In the coronary artery, NTG (1-100 nM) produced relaxations with EC50 = 9.4 nM. In KCl-contracted arteries (20-80 mM KCl), relaxation by NTG was progressively reduced. Relaxation responses to NTG also were inhibited significantly by potent calcium-activated K+ (BK) channel blockers, charybdotoxin (100 nM) and iberiotoxin (200 nM), but not by KATP blockers such as PNU-37883A (10 microM) or PNU-99963 (100 nM). Nitric oxide (0.1-30 nM) and acetylcholine (3-300 nM) also produced relaxations which were significantly attenuated by the BK blockers. In further experiments, NTG (1-100 nM) produced inhibition of PNU-46619-induced SR [Ca++]i release, with an IC50 of 8.5 nM, which was not affected by charybdotoxin. Furthermore, P1075 (50 nM), a KATP opener, did not inhibit agonist-stimulated SR [Ca++]i release. Ryanodine (10 microM), which acts on SR Ca++ release channels, did not alter NTG relaxations, whereas thapsigargin (0.1 microM), a selective inhibitor of SR Ca(++)-ATPase pump, produced pronounced inhibition of NTG relaxations. These results suggest that NTG, in the therapeutic concentration range, produces coronary relaxation primarily via two cellular mechanisms: plasmalemmal BK channel activation and stimulation of SR Ca(++)-ATPase to produce increased SR Ca++ accumulation. These two mechanisms apparently are equally important and act together to produce a unique vasorelaxation profile demonstrated by NTG-type coronary vasodilators.

Topics: Animals; Calcium; Charybdotoxin; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Male; Nitroglycerin; Peptides; Potassium; Potassium Channel Blockers; Potassium Channels; Ryanodine; Thapsigargin; Vasodilation; Vasodilator Agents

1. The effects of adrenergic agonists on K+ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells. 2. Outward K+ current (IK) was reduced by tetraethylammonium chloride, the Ca2+-activated K+ (K(Ca)) channel blocker iberiotoxin (IbTX), or Ca2+-free external Ringer solution. The calcium ionophore ionomycin increased an IbTX-sensitive IK in PCE cells. 3. The adrenergic agonists adrenaline and phenylephrine increased IK in PCE cells. The induced current was blocked by IbTX and the alpha1-antagonist prazosin, suggesting that adrenergic agonists activate IK(Ca) via alpha1-adrenoreceptors. 4. Internal dialysis of D-myo-inositol 1,4, 5-trisphosphate (IP3) increased IK, whilst pre-incubation of PCE cells with thapsigargin or the phospholipase C (PLC) inhibitor U-73122 reduced phenylephrine-induced increases in IK(Ca). Adrenergic increases in IK(Ca) were mediated by a pertussis toxin-insensitive G protein. 5. These results demonstrate that IK(Ca) channels in rabbit PCE cells are coupled to alpha1-adrenergic receptors and a PLC/IP3 signalling pathway. Activation of these channels may modulate fluid secretion by the ciliary epithelium.

Topics: Adrenergic alpha-Agonists; Animals; Calcium-Transporting ATPases; Cells, Cultured; Ciliary Body; Enzyme Inhibitors; Epinephrine; Epithelial Cells; Estrenes; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Ionomycin; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Peptides; Phenylephrine; Potassium Channels; Potassium Channels, Calcium-Activated; Prazosin; Pyrrolidinones; Rabbits; Receptors, Adrenergic, alpha-1; Scorpion Venoms; Signal Transduction; Tetraethylammonium; Thapsigargin; Type C Phospholipases

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 Ca(2+)-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.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Cadmium; Calcium; Calcium Channel Agonists; Calcium Channels; Cell Membrane; Cerebral Arteries; Membrane Potentials; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Peptides; Potassium Channels; Rats; Ryanodine; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Vasodilation