RINm5f cells express inactivating BK channels whereas HIT cells express noninactivating BK channels. (1/460)

Large-conductance Ca2+- and voltage-activated BK-type K+ channels are expressed abundantly in normal rat pancreatic islet cells and in the clonal rat insulinoma tumor (RINm5f) and hamster insulinoma tumor (HIT) beta cell lines. Previous work has suggested that the Ca2+ sensitivity of BK channels in RIN cells is substantially less than that in HIT cells, perhaps contributing to differences between the cell lines in responsiveness to glucose in mediating insulin secretion. In both RIN cells and normal pancreatic beta cells, BK channels are thought to play a limited role in responses of beta cells to secretagogues and in the electrical activity of beta cells. Here we examine in detail the properties of BK channels in RIN and HIT cells using inside-out patches and whole cell recordings. BK channels in RIN cells exhibit rapid inactivation that results in an anomalous steady-state Ca2+ dependence of activation. In contrast, BK channels in HIT cells exhibit the more usual noninactivating behavior. When BK inactivation is taken into account, the Ca2+ and voltage dependence of activation of BK channels in RIN and HIT cells is essentially indistinguishable. The properties of BK channel inactivation in RIN cells are similar to those of inactivating BK channels (termed BKi channels) previously identified in rat chromaffin cells. Inactivation involves multiple, trypsin-sensitive cytosolic domains and exhibits a dependence on Ca2+ and voltage that appears to arise from coupling to channel activation. In addition, the rates of inactivation onset and recovery are similar to that of BKi channels in chromaffin cells. The charybdotoxin (CTX) sensitivity of BKi currents is somewhat less than that of the noninactivating BK variant. Action potential voltage-clamp waveforms indicate that BK current is activated only weakly by Ca2+ influx in RIN cells but more strongly activated in HIT cells even when Ca2+ current magnitude is comparable. Concentrations of CTX sufficient to block BKi current in RIN cells have no effect on action potential activity initiated by glucose or DC injection. Despite its abundant expression in RIN cells, BKi current appears to play little role in action potential activity initiated by glucose or DC injection in RIN cells, but BK current may play an important role in action potential repolarization in HIT cells.  (+info)

Calcium responses induced by acetylcholine in submucosal arterioles of the guinea-pig small intestine. (2/460)

1. Calcium responses induced by brief stimulation with acetylcholine (ACh) were assessed from the fluorescence changes in fura-2 loaded submucosal arterioles of the guinea-pig small intestine. 2. Initially, 1-1.5 h after loading with fura-2 (fresh tissues), ACh increased [Ca2+]i in a concentration-dependent manner. This response diminished with time, and finally disappeared in 2-3 h (old tissues). 3. Ba2+ elevated [Ca2+]i to a similar extent in both fresh and old tissues. ACh further increased the Ba2+-elevated [Ca2+]i in fresh tissues, but reduced it in old tissues. Responses were not affected by either indomethacin or nitroarginine. 4. In fresh mesenteric arteries, mechanical removal of endothelial cells abolished the ACh-induced increase in [Ca2+]i, with no alteration of [Ca2+]i at rest and during elevation with Ba2+. 5. In the presence of indomethacin and nitroarginine, high-K+ solution elevated [Ca2+]i in both fresh and old tissues. Subsequent addition of ACh further increased [Ca2+]i in fresh tissues without changing it in old tissues. 6. Proadifen, an inhibitor of the enzyme cytochrome P450 mono-oxygenase, inhibited the ACh-induced changes in [Ca2+]i in both fresh and Ba2+-stimulated old tissues. It also inhibited the ACh-induced hyperpolarization. 7. In fresh tissues, the ACh-induced Ca2+ response was not changed by apamin, charybdotoxin (CTX), 4-aminopyridine (4-AP) or glibenclamide. In old tissues in which [Ca2+]i had previously been elevated with Ba2+, the ACh-induced Ca2+ response was inhibited by CTX but not by apamin, 4-AP or glibenclamide. 8. It is concluded that in submucosal arterioles, ACh elevates endothelial [Ca2+]i and reduces muscular [Ca2+]i, probably through the hyperpolarization of endothelial or smooth muscle membrane by activating CTX-sensitive K+ channels.  (+info)

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve. (3/460)

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.  (+info)

Differences in the actions of some blockers of the calcium-activated potassium permeability in mammalian red cells. (4/460)

1. The actions of some inhibitors of the Ca2+-activated K+ permeability in mammalian red cells have been compared. 2. Block of the permeability was assessed from the reduction in the net loss of K+ that followed the application of the Ca2+ ionophore A23187 (2 microM) to rabbit red cells suspended at a haematocrit of 1% in a low potassium solution ([K]0 0.12-0.17 mM) at 37 degrees C. Net movement of K+ was measured using a K+-sensitive electrode placed in the suspension. 3. The concentrations (microM +/- s.d.) of the compounds tested causing 50% inhibition of K+ loss were: quinine, 37 +/- 3; cetiedil, 26 +/- 1; the cetiedil congeners UCL 1269, UCL 1274 and UCL 1495, approximately 150, 8.2 +/- 0.1, 0.92 +/- 0.03 respectively; clotrimazole, 1.2 +/- 0.1; nitrendipine, 3.6 +/- 0.5 and charybdotoxin, 0.015 +/- 0.002. 4. The characteristics of the block suggested that compounds could be placed in two groups. For one set (quinine, cetiedil, and the UCL congeners), the concentration-inhibition curves were steeper (Hill coefficient, nH, > or = 2.7) than for the other (clotrimazole, nitrendipine, charybdotoxin) for which nH approximately 1. 5. Compounds in the first set alone became less active on raising the concentration of K+ in the external solution to 5.4 mM. 6. The rate of K+ loss induced by A23187 slowed in the presence of high concentrations of cetiedil and its analogues, suggesting a use-dependent component to the inhibitory action. This was not seen with clotrimazole. 7. The blocking action of the cetiedil analogue UCL 1274 could not be overcome by an increase in external Ca2+ and its potency was unaltered when K+ loss was induced by the application of Pb2+ (10 microM) rather than by A23187. 8. These results, taken with the findings of others, suggest that agents that block the red cell Ca2+-activated K+ permeability can be placed in two groups with different mechanisms of action. The differences can be explained by supposing that clotrimazole and charybdotoxin act at the outer face of the channel whereas cetiedil and its congeners may block within it, either at or near the K+ binding site that determines the flow of K+.  (+info)

Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line. (5/460)

1. Short-circuit current (I(SC)) responses to carbachol (CCh) were investigated in Colony 1 epithelia, a subpopulation of the HCA-7 adenocarcinoma cell line. In Krebs-Henseleit (KH) buffer, CCh responses consisted of three I(SC) components: an unusual rapid decrease (the 10 s spike) followed by an upward spike at 30 s and a slower transient increase (the 2 min peak). This response was not potentiated by forskolin; rather, CCh inhibited cyclic AMP-stimulated I(SC). 2. In HCO3- free buffer, the decrease in forskolin-elevated I(SC) after CCh was reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl- anions were replaced by gluconate, both Ca2+- and cyclic AMP-mediated electrogenic responses were significantly inhibited. 3. Basolateral Ba2+ (1-10 mM) and 293B (10 microM) selectively inhibited forskolin stimulation of I(SC), without altering the effects of CCh. Under Ba2+- or 293B-treated conditions, CCh responses were potentiated by pretreatment with forskolin. 4. Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3- free medium, without affecting the 2 min peak. The enhanced 10 s spike was inhibited by prior addition of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses. 5. In epithelial layers prestimulated with forskolin, the muscarinic antagonists atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, both at 100 nM) abolished subsequent 10 microM CCh responses. Following addition of p-fluoro hexahydro-sila-difenidol (pF-HHSiD, 10 microM) or pirenzepine (1 microM), qualitative changes in the CCh response time-profile also indicated a rightward shift of the agonist concentration-response curve; however, 1 microM gallamine had no effect. These results suggest that a single M3-like receptor subtype mediates the secretory response to CCh. 6. It is concluded that CCh and forskolin activate discrete populations of basolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl- permeability of the apical membrane may limit their combined effects on electrogenic Cl- secretion. In addition, CCh activates a Ba2+-sensitive apical K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3- secretion through a mechanism at least partially dependent on carbonic anhydrase.  (+info)

Mechanical stimulation regulates voltage-gated potassium currents in cardiac microvascular endothelial cells. (6/460)

Vascular endothelial cells are constantly exposed to mechanical forces resulting from blood flow and transmural pressure. The goal of this study was to determine whether mechanical stimulation alters the properties of endothelial voltage-gated K+ channels. Cardiac microvascular endothelial cells (CMECs) were isolated from rat ventricular muscle and cultured on thin sheets of silastic membranes. Membrane currents were measured with the use of the whole-cell arrangement of the patch-clamp technique in endothelial cells subjected to static stretch for 24 hours and compared with measurements from control, nonstretched cells. Voltage steps positive to -30 mV resulted in the activation of a time-dependent, delayed rectifier K+current (IK) in the endothelial cells. Mechanically induced increases of 97%, 355%, and 106% at +30 mV were measured in the peak amplitude of IK in cells stretched for 24 hours by 5%, 10%, and 15%, respectively. In addition, the half-maximal voltage required for IK activation was shifted from +34 mV in the nonstretched cells to -5 mV in the stretched cells. Although IK in both groups of CMECs was blocked to a similar extent by tetraethylammonium, currents in the stretched endothelial cells displayed an enhanced sensitivity to inhibition by charybdotoxin. Preincubation of the CMECs with either pertussis toxin or phorbol 12-myristate 13-acetate during the 24 hours of cell stretch did not prevent the increase in IK. The application of phorbol 12-myristate 13-acetate and static stretch stimulated the proliferation of CMECs. Stretch-induced regulation of K+ channels may be important to control the resting potential of the endothelium and may contribute to capillary growth during periods of mechanical perturbation.  (+info)

Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. (7/460)

In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated (n = 5) or myogenic tone (n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.  (+info)

Differential effects of pinacidil, cromakalim, and NS 1619 on electrically evoked contractions in rat vas deferens. (8/460)

AIM: To compare the inhibitory action of electrically evoked contractions of rat epididymal vas deferens by pinacidil (Pin), cromakalim (Cro), and NS 1619. METHODS: Monophasic contractions were evoked by electric field stimulation in rat isolated epididymal half of vas deferens. RESULTS: Newly developed ATP-sensitive K+ channel openers, Pin and Cro, concentration-dependently reduced the electrically evoked (0.3 Hz, 1 ms pulse duration, 60 V) contractions and glibenclamide but not charybdotoxin antagonized the inhibitory effects of both agents. Pin shifted the concentration-response curve for norepinephrine to the right with reducing the magnitude of the maximum contraction in a glibenclamide-sensitive fashion. The large-conductance Ca(2+)-activated K+ channel opener, NS 1619, inhibited the electrically evoked contractions in a concentration-dependent manner. Charybdotoxin (100 nmol.L-1) partially reduced the effect of NS 1619 but glibenclamide (10 mumol.L-1) showed no effect. None of these 3 agents affected the basal tension. CONCLUSION: Both ATP-sensitive and Ca(2+)-activated K+ channels presented in vas deferens smooth muscles involved in regulation of muscle contractility.  (+info)

• 1
• 2
• 3
• 4
• 5
• 6
• 7
• 8
• 9
• 10