Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Calcium transport proteins in the nonfailing and failing heart: gene expression and function

In heart failure alterations of intracellular Ca²⁺ handling are thought to be a major reason for impaired contraction and relaxation. Peak Ca²⁺ transients are reduced, resting Ca²⁺ levels elevated, and the time course of diastolic Ca²⁺ decline is markedly prolonged in failing hearts. The proteins of the sarcoplasmic reticulum and the sarcolemmal Na⁺/Ca²⁺ exchanger are the most important tools for Ca²⁺ homeostasis in the cardiomyocyte, and their molecular cloning has allowed prediction of structure/function analysis. The investigation of function and gene expression of these proteins in failing myocardium has been an area of intensive research in recent years in order to provide a more detailed understanding of the pathophysiology of heart failure. Quantitative changes in expression of the sarcoplasmic reticulum Ca²⁺-ATPase, the ryanodine receptor, and the Na⁺/Ca²⁺ exchanger with correlations to functional alterations have been reported both in experimental animal models and in the human failing heart. However, in human heart failure these findings are currently the subject of a lively discussion because observations have apparently been in part contradictory. This review discusses the proteins involved in myocardial Ca²⁺ handling and describes the current state of research on expressional and functional alterations and their potential implication in the pathomechanism of heart failure.Abbreviations ANF Atrial natriuretic factor - PLN Phospholamban - RyR Ryanodine receptor - SR Sarcoplasmic reticulum - SERCA Sarco(endo)plasmic reticulum Ca²⁺-ATPase     

Activity-dependent intracellular Ca2+ transients in unmyelinated nerve fibres of the isolated adult rat vagus nerve

 Confocal laser scanning microscopy was used to follow changes in the free intracellular calcium concentration ([Ca²⁺]_i) in nerve fibres and adjacent Schwann cells in isolated rat vagus nerves. [Ca²⁺]_i was monitored by the Ca²⁺-sensitive fluorescent dyes Calcium Green-1 and Fura Red. Intracellular Ca²⁺ transients were observed during repetitive (1–50 Hz) supramaximal electrical stimulation or by bath application of ATP. Trains of action potentials were more effective at elongated, fibre-like structures of the vagus nerves, whereas ATP-induced Ca²⁺ transients were found predominantly in regions of Schwann cell bodies. Activity-induced Ca²⁺ signals were unaffected by pharmacological manipulation of intracellular Ca²⁺ stores, during long-lasting application of purinergic receptor agonists, or by substitution of extracellular Na⁺ with Li⁺. However, they were abolished in the presence of Ca²⁺-free bathing solution or after the blocking of Ca²⁺ channels with Cd²⁺. Ca²⁺ transients were also observed during Ca²⁺ action potentials. Such ”Ca²⁺ spikes” were elicited by electrical stimulation in the presence of a combination of tetrodotoxin and K⁺ channel blockers. These data suggest that voltage-dependent Ca²⁺ channels, activated during short trains of Na⁺ action potentials, produce an increase in intra-axonal [Ca²⁺] of rat vagus nerves. We did not find evidence for activity-dependent Ca²⁺ transients in the Schwann cells surrounding the unmyelinated axons. Received: 16 September 1997 / Received after revision: 25 November 1997 / Accepted: 27 November 1997     

Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate – and HEPES‐buffered solutions

Aim: The aim of this study was to compare the action potential configuration, contractility, intracellular Ca²⁺ and H⁺ concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 °C. Results: In Langendorff‐perfused guinea‐pig hearts, loaded with the fluorescent Ca²⁺‐indicator Fura‐2, or H⁺‐sensitive dye carboxy‐SNARF, shifts from Krebs to Tyrode solution caused intra‐cellular acidification, increased diastolic pressure and [Ca²⁺]_i, decreased systolic pressure and [Ca²⁺]_i, leading to a reduction in the amplitude of [Ca²⁺]_i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca²⁺]_i and [H⁺]_i were readily reversible by addition of 26 mmol L^?1 bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. Conclusion: It is concluded that the marked differences in action potential morphology, [Ca²⁺]_i, [H⁺]_i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.     

Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+

Aim: We have investigated the influence of Ca²⁺ ions on the basic biophysical properties of T‐type calcium channels. Methods: The Ca_v3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca²⁺ or Na⁺ ions were used as charge carriers. The intracellular Ca²⁺ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 μm Ca²⁺ into the non‐buffered intracellular solution. Various combinations of extra‐ and intracellular solutions yielded high, intermediate or low intracellular Ca²⁺ levels. Results: The amplitude of the calcium current was independent of intracellular Ca²⁺ concentrations. High levels of intracellular Ca²⁺ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca²⁺ by Na⁺ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca²⁺ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ?100 mV for the Ca²⁺ current, but did not reach steady state even at ?150 mV for the Na⁺ current. Conclusions: Ca²⁺ ions facilitate transitions of Ca_v3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.     

Effect of activation of protein kinase C on excitation-contraction coupling in frog twitch muscle fibres

Intracellular Ca²⁺ transients were recorded from frog twitch muscle fibres in response to voltage-clamp depolarizing pulses, using arsenazo III as an intracellular Ca²⁺ indicator. The effect of the activation of protein kinase C (PKC) on the Ca²⁺ transients was studied. With 1 M phorbol 12,13-dibutyrate (PDBu), a PKC activator, the peak of the Ca²⁺ transients increased to about 120% of control during the first 0.5 h, and then decreased gradually to a plateau of 44% of control within the following 2 h. This effect of PDBu could be alleviated significantly by PKC inhibitors, 10 M polymyxin B (PMB) or 30 M 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7). Moreover, PDBu caused an upward shift of the strength/duration curve. In Li⁺-loaded muscle fibres the Ca²⁺ transients could not fully recover after 80 mM K⁺ exposure for 15 min, while the post-K⁺ Ca²⁺ transients could be completely restored in the fibres not loaded with Li⁺. In the presence of 10 M PMB or 30 M H-7, a full restoration of the post-K⁺ Ca²⁺ transients was seen in Li⁺-loaded fibres. PMB supplemented after high-K⁺ exposure also could result in a complete recovery of the post-K⁺ Ca²⁺ transients in Li⁺-loaded fibres. The role of PKC in modulating excitation-contraction coupling in frog twitch muscle fibres is clearly indicated, but the mechanism(s) and physiological significance remain to be established.     

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca²⁺-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing α-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively α-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, α-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than β-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.     

Renal sodium/calcium exchange; a vasodilator that is defective in salt‐sensitive hypertension

The Na⁺ : Ca²⁺ exchanger is an important plasma membrane ion transport pathway that plays a major role in controlling [Ca²⁺]_i. In smooth muscle cells, it may function as a Ca²⁺ extrusion pathway and may help lower [Ca²⁺]_i in response to vasoconstrictor‐induced increases in [Ca²⁺]_i. It may also extrude [Ca²⁺]_i and lead to vasodilation in response to vasodilators. Our recent studies have been performed to determine the existence and regulation of the Na⁺ : Ca²⁺ exchanger in renal contractile cells which include afferent and efferent arterioles and mesangial cells. Exchanger activity is present in all three of these contractile elements but is higher in afferent arterioles vs. efferent arterioles. We have also examined the role of altered regulation of the exchanger in the SHR and in salt‐sensitive hypertension. With the establishment of high blood pressure, Na⁺ : Ca²⁺ exchanger activity is reduced in afferent but not in efferent arterioles in both models of hypertension. Other works in cultured mesangial cells and freshly dissected afferent arterioles, have shown that protein kinase C (PKC) up‐regulates the Na⁺ : Ca²⁺ exchanger from Dahl/Rapp salt‐resistant rats while it fails to do so in arterioles and mesangial cells from salt‐sensitive rats. This defect in PKC regulation of Na⁺ : Ca²⁺ exchange is the result of a loss of PKC‐mediated translocation of the exchanger to the plasma membrane in S mesangial cells. Thus, a defect in the PKC‐Na⁺ : Ca²⁺ exchanger‐translocation pathway may cause dysregulation of [Ca²⁺]_i and help explain the dramatic decrease in GFR that occurs in this model of hypertension.     

Late sodium current contributes to diastolic cell Ca<Superscript>2+</Superscript> accumulation in chronic heart failure

We elucidate the role of late Na⁺ current (I_NaL) for diastolic intracellular Ca²⁺ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca²⁺ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). I_NaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca²⁺ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca²⁺ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective I_NaL blocker ranolazine (10 μM) or the prototypical Na⁺ channel blocker tetrodotoxin (2 μM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of I_NaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that I_NaL contributes to diastolic Ca²⁺ accumulation and spontaneous Ca²⁺ release in HF.     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Osmolality- and Na+-dependent effects of hyperosmotic NaCl solution on contractile activity and Ca2+ cycling in rat ventricular myocytes

Hypertonic NaCl solutions have been used for small-volume resuscitation from hypovolemic shock. We sought to identify osmolality- and Na⁺-dependent components of the effects of the hyperosmotic NaCl solution (85 mOsm/kg increment) on contraction and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in isolated rat ventricular myocytes. The biphasic change in contraction and Ca²⁺ transient amplitude (decrease followed by recovery) was accompanied by qualitatively similar changes in sarcoplasmic reticulum (SR) Ca²⁺ content and fractional release and was mimicked by isosmotic, equimolar increase in extracellular [Na⁺] ([Na⁺]_o). Raising osmolality with sucrose, however, augmented systolic [Ca²⁺]_i monotonically without change in SR parameters and markedly decreased contraction amplitude and diastolic cell length. Functional SR inhibition with thapsigargin abolished hyperosmolality effects on [Ca²⁺]_i. After 15-min perfusion, both hyperosmotic solutions slowed mechanical relaxation during twitches and [Ca²⁺]_i decline during caffeine-evoked transients, raised diastolic and systolic [Ca²⁺]_i, and depressed systolic contractile activity. These effects were greater with sucrose solution, and were not observed after isosmotic [Na⁺]_o increase. We conclude that under the present experimental conditions, transmembrane Na⁺ redistribution apparently plays an important role in determining changes in SR Ca²⁺ mobilization, which markedly affect contractile response to hyperosmotic NaCl solutions and attenuate the osmotically induced depression of contractile activity.     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Mild stress of caffeine increased mtDNA content in skeletal muscle cells: the interplay between Ca2+ transients and nitric oxide

Caffeine increases mitochondrial biogenesis in myotubes by evoking Ca²⁺ transients. Nitric oxide (NO) also induces mitochondrial biogenesis in skeletal muscle cells via upregulation of AMP-activated protein kinase (AMPK) activity and PGC-1??. However, the interplay and timing sequence between Ca²⁺ transients and NO releases remain unclear. Herein, we tested the hypothesis that caffeine-evoked Ca²⁺ transients triggered NO production to increase mtDNA in skeletal muscle cells. Ca²⁺ transients were recorded with Fura-2 AM and confocal microscopy; mtDNA staining, mitochondrial membrane potential and NO level were determined using fluorescent probes PicoGreen, tetramethylrhodamine methyl ester (TMRM) and DAF-FM, respectively. In primary cultured myotubes, a subtle and moderate stress of caffeine increased mtDNA exclusively. Mitochondrial membrane potential and mtDNA were increased by 1?mM as well as 5?mM caffeine, whereas 10?mM caffeine did not change the fluorescence intensity of PicoGreen and TMRM. NO level in myocytes increased gradually following the first jump of Ca²⁺ transients evoked by caffeine (5?mM) till the end of recording, when Fura-2 indicated that Ca²⁺ transients recovered partly and even disappeared. Importantly, nitric oxide synthase (NOS) inhibitor (l-NAME) suppressed caffeine-induced mtDNA biogenesis, whereas NO donor (DETA-NO) increased mtDNA content. These data strongly suggest that caffeine-induced mtDNA biogenesis is dose-sensitive and dependent on a certain level of stress. Further, an increasing level of NO following Ca²⁺ transients is required for caffeine-induced mtDNA biogenesis. Additionally, Ca²⁺ transients, a usual and first response to caffeine, was either suppressed or attenuated by l-NAME, DETA-NO, AICAR and U0126, suggesting an inability to control [Ca²⁺]_i in these treated cells. There may be an important interplay between NO and Ca²⁺ transients in intracellular signaling system involving NOS, AMPK and MEK.     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea‐pig urinary bladder smooth muscle

In the present study, effects of different types of K⁺ channel modulators on the spontaneous rhythmic contractile activity were examined in guinea‐pig urinary bladder smooth muscle (UBSM). Guinea‐pig UBSM exhibited myogenic rhythmic contraction in the presence of atropine (1 μM ), phentolamine (1 μM ), propranolol (1 μM ), suramin (10 μM ) and tetrodotoxin (1 μM ). Nisoldipine (100 nM ) or diltiazem (10 μM ) substantially diminished UBSM contractile activity. Nisoldipine‐resistant component of UBSM rhythmic contraction was further inhibited by gadolinium (200 μM ). Iberiotoxin (50 nM ), a selective blocker of large‐conductance, voltage‐gated Ca²⁺‐activated K⁺ (K_Ca) (BK) channel, dramatically increased both contraction amplitude and frequency whereas NS‐1619 (30 μM ), which increases BK channel activity, decreased them. Apamin (100 nM ), a selective blocker of small‐conductance, K_Ca (SK) channel, increased contraction amplitude but decreased frequency. A blocker of voltage‐gated K⁺ (K_v) channel, 4‐aminopyridine (100 μM ), significantly increased contraction frequency. E‐4031, a blocker of a novel inwardly rectifying K⁺ channel, i.e. the human ether‐a‐go‐go‐related gene (HERG) K⁺ channel, significantly increased contraction amplitude. Glibenclamide (1–10 μM ) (K_ATP channel blocker) and Ba²⁺ (10 μM ) (conventional K_ir channel blocker) did not exhibit conspicuous effects on spontaneous contractile activity of UBSM. These findings imply that two types of K_Ca (BK and SK) channels have prominent roles as negative feedback elements to limit extracellular Ca²⁺ influx‐mediated guinea‐pig UBSM contraction by regulating both amplitude and frequency. It was also suggested that both non‐K_Ca type of K⁺ (K_v and HERG‐like K⁺) channels may contribute to the regulation of UBSM myogenic rhythmic contraction.