Summary
We investigated two putative K+ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K+, tetraethylammonium (TEA), or norepinephrine. The K+ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K+. Measurements of intracellular free Ca++ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca++-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca++ concentrations, called hot spots, were determined by a computer-generated, 3λ algorithm. Hot spots, which most likely represent the sites of Ca++ release and re-uptake by Ca++-regulatory organelles, increased in size and intensity upon addition of K+ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca++ activity. Both norepinephrine and K+-induced stimulation resulted in Ca++ activity increases that were primarily due to Ca++ release from storage sites. Reduction of free Ca++ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca++ decreases due to K+ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca++ activity while elevated Ca++ levels persisted at the periphery. While this late stage residual of peripheral Ca++ appears to be a significant step in the vascular muscle relaxant action of both K+ channel opener drugs, the level of Ca++ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K+ conductance, pinacidil and cromakalim cause 1) decreased Ca++ activity in central regions of the myocytes, and 2) a shift in Ca++ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca++ activity within a vascular muscle cell, with Ca++ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca++ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K+ channel openers.
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References
Aprigliano O, Hermsmeyer K (1976) In vitro denervation of the portal vein and caudal artery of the rat. J Pharmacol Exp Ther 198:578–587
Bond M, Kitazawa T, Shuman H, Somlyo AV, Somlyo AP (1984a) Ca++ release from and recycling by the sarcoplasmic reticulum in guinea-pig smooth muscle. J Physiol 355:677–695
Bond M, Shuman H, Somlyo AP, Somlyo AV (1984b) Total cytoplasmic Ca++ in relaxed and maximally contracted rabbit portal vein smooth muscle. J Physiol 357:185–201
Bright GR, Fisher GW, Rogowska J, Taylor DL (1987) Fluorescence ratio imaging microscopy: Temporal and spatial measurements of cytoplasmic pH. J Cell Biol 104:1019–1033
Clapham JC, Wilson C (1987) Anti-spasmogenic and spasmolytic effects of BRL34915: a comparison with nifedipine and nicorandil. J Auton Pharmacol 7:233–242
Cook NS (1987) The pharmacology of potassium channels and their therapeutic potential. TIPS 9:21–28
Cook NS, Hof RP (1988) Cardiovascular effects of apamin and BRL34915 in rats and rabbits. Br J Pharmacol 93:121–131
Cook NS, Quast U, Hof RP, Baumlin Y, Pally C (1988) Similarities in the mechanism of action of two new vasodilator drugs: pinacidil and BRL34915. J Cardiol Pharmacol 11:90–99
Duggleby RG (1981) A nonlinear regression program for small computers. Anal Biochem 110:9–18
Erne P, Hermsmeyer K (1988a) Intracellular Ca++ release in vascular muscle cells by caffeine, ryanodine, norepinephrine, and neuropeptide Y. J Cardiovasc Pharmacol 12 [Suppl 5]:S85-S91
Erne P, Hermsmeyer K (1988b) Desensitization to norepinephrine includes refractoriness of calcium release in myocardial cells. Biochem Biophys Res Commun 151:333–338
Erne P, Hermsmeyer K (1989) Intracellular vascular muscle calcium modulation in genetic hypertension. Hypertension 14:145–151
Erne P, Bolli P, Bürgisser E, Bühler FR (1984a) Correlation of platelet calcium with blood pressure. N Engl J Med 310:1084–1088
Erne P, Mittelholzer E, Bürgisser E, Flückiger R, Bühler FR (1984b) Measurement of the receptor induced changes in intracellular free calcium in human platelets. J Receptor Res 4:587–604
Erne P, Kiowski W, Resink TJ, Hefti A, Ritz R, Bühler FR (1985) Hormonal modulation of intracellular free calcium in platelets from normotensive and hypertensive subjects. Klin Wochenschr 63 [Suppl 3]:111–112
Erne P, Chiesi M, Longoni S, Fulbright J, Hermsmeyer K (1989) Relaxation of rat vascular muscle by peripheral benzodiazepine modulators. J Clin Invest 84:493–498
Escande D, Thuringer D, Leguern S, Cavero I (1988) The potassium channel opener cromakalim (BRL34915) activates ATP-dependent K+ channels in isolated cardiac myocytes. Biochem Biophys Res Commun 154:620–625
Goldman WF, Wier WG, Blaustein MP (1989) Effects of activation on distribution of Ca++ in single arterial smooth muscle cells. Circ Res 64:1019–1029
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca++ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3449
Haeusler G (1983) Contraction, membrane potential, and calcium fluxes in rabbit pulmonary arterial muscle. Fed Proc 42:672–682
Hermsmeyer K (1976) Ba++ and K+ alteration of K+ conductance in spontaneously active vascular muscle. Am J Physiol 230:1031–1036
Hermsmeyer K (1979) High shortening velocity of isolated single arterial muscle cells. Experientia 35:1599–1602
Hermsmeyer K (1983) Sodium pump hyperpolarization-relaxation in rat caudal artery. Fed Proc 42:246–252
Hermsmeyer K (1988a) Pinacidil actions on ion channels in vascular muscle. J Cardiovasc Pharmacol [Suppl 2]:S17-S22
Hermsmeyer K (1988b) Ion channel effects of pinacidil in vascular muscle. Drugs 36:29–32
Hermsmeyer K, Akbarali H (1989) Cellular pacemaker mechanism in vascular muscle. Prog Appl Microcirc 15:32–40
Hermsmeyer K, Mason R (1982) Norepinephrine sensitivity and desensitization of cultured single vascular muscle cells. Circ Res 50:627–632
Hermsmeyer K, Robinson R (1977) High sensitivity of cultured cardiac muscle cells to autonomic agents. Am J Physiol 233:C172-C179
Highsmith S, Bloebaum P, Snowdowne KW (1986) Sarcoplasmic reticulum interacts with calcium indicator precursor fura2 AM. Biochem Biophys Res Comm 138:1153–1162
Inoue R, Okabe K, Kitamura K, Kuriyama H (1985) Two Ca-dependent K-channels classified by the application of tetraethylammonium distribute to smooth muscle membranes of the rabbit portal vein. Pflügers Arch 405:173–179
Jackson AP, Timmerman MP, Bagshaw CR, Ashley CC (1987) The kinetics of calcium binding to fura2 and indo1. FEBS 216:35–39
Kakei M, Yoshinaga M, Saito K, Tanaka H (1986) The potassium current activated by 2-nicotinamidoethyl nitrate (nicorandil) in single ventricular cells of guinea pigs. Proc Royal Soc Lond 229:331–343
Klockner U, Trieschmann U, Isenberg G (1989) Pharmacological modulation of calcium and potassium channels in isolated vascular smooth muscle cells. Arzneim-Forsch/Drug Res 39:120–126
Kowarski D, Shuman H, Somlyo AP, Somlyo AV (1985) Calcium release by noradrenaline from central sarcoplasmic reticulum in rabbit main pulmonary artery smooth muscle. J Physiol 366:153–175
Kwan CY, Takemura H, Obie JF, Thastrup O, Putney JW Jr (1990) Effects of McCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. Am J Physiol 258 (Cell Physiol 27):C1006-C1015
Marvin W, Robinson R, Hermsmeyer K (1979) Correlation of function and morphology of neonatal rat and embryonic chick cultured cardiac and vascular muscle cells. Circ Res 45:528–540
Osterrieder W (1988) Modification of K+ conductance of heart cell membrane by BRL34915. Naunyn-Schmiedeberg's Arch Pharmacol 337:93–97
Post JM, Smith JM, Jones AW (1989) BRL34915 (Cromakalim) stimulation of 42K efflux from rabbit arteries is modulated by calcium. J Pharmacol Exp Ther 250:591–597
Quast U, Baumlin Y (1988) Comparison of the effluxes of 42K and 86Rb+ elicited by cromakalim (BRL34915) in tonic and phasic vascular tissue. Naunyn-Schmiedeberg's Arch Pharmacol 338:319–326
Scholtysik G (1987) Evidence for inhibition by ICS 205–930 and stimulation by BRL34915 of K+ conductance in cardiac muscle. Naunyn-Schmiedeberg's Arch Pharmacol 335:692–696
Shetty SS, Weiss GB (1987) Dissociation of actions of BRL34915 in the rat portal vein. Eur J Pharmacol 141:485–488
Sturek M, Hermsmeyer K (1986) Calcium and sodium channels in spontaneously contracting vascular muscle cells. Science 233:475–478
Tsien RY, Pozzan T, Rink TJ (1983) Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new intracellularly trapped fluorescent indicator. J Cell Biol 94:325–334
van Breemen C, Leuten P, Yamamoto H, Aaronson P, Cauvin C (1986a) Calcium activation of vascular smooth muscle. [State of the art lecture.] Hypertension 8 [Suppl II]:85–95
van Breemen C, Lukeman S, Leijten P, Yamamoto H, Loutzenhiser R (1986b) The role of superficial SR in modulating force development induced by Ca++ entry into arterial smooth muscle. J Cardiovasc Pharmacol 8 [Suppl8]:S111-S116
Videbaek LM, Aalkjaer C, Hughes AD, Mulvany MJ (1990) Effect of pinacidil on ion permeability in resting and contracted resistance vessels. Am J Physiol 259 (Heart Cite Physiol 28):H14-H22
Wick RA (1987) Quantum-limited imaging using microchannel plate technology. Appl Optics 26:3210–3218
Wier WG, Cannel MB, Berlin JR, Marban E, Lederer WJ (1987) Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura2. Science 235:325–328
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This study was supported by NIH grants HL38537 and HL38645, and Eli Lilly Co. P.E. was supported by the Swiss Foundation of Cardiology and by the SNF 32-029 975.90
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Erne, P., Hermsmeyer, K. Modulation of intracellular calcium by potassium channel openers in vascular muscle. Naunyn-Schmiedeberg's Arch Pharmacol 344, 706–715 (1991). https://doi.org/10.1007/BF00174755
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DOI: https://doi.org/10.1007/BF00174755