High Potassium Solution Research Articles

Calcium-Antagonistic Activity of Sumatriptan in the Rat Anococcygeus Muscle

The relaxant effect of sumatriptan, a 5-HT_1B/1D receptor agonist, on the rat anococcygeus muscle was investigated. Sumatriptan induced concentration-dependent relaxations of the phenylephrine-precontracted rat anococcygeus muscle. N^G-nitro-L-arginine, methylene blue, glibenclamide, tetrodotoxin, indomethacin, GR 113808, GR 55562, methysergide, ketanserin, ICS 205930, clozapine, methiothepin, metergoline, mesulergine, and ritanserin did not inhibit the relaxations induced by sumatriptan. Sumatriptan converted calcium-induced contractions into relaxations in the preparations depolarized by a calcium-free, high-potassium solution. In membrane preparations obtained from the rat anococcygeus muscle, the basal rate of cAMP production by adenylate cyclase was 4.77 ± 0.02 pmol/mg protein/min (n = 15). The enzyme activity was increased to 104 ± 51.7 pmol/mg protein/min by forskolin (10^–4 mol/l), but did not change in the presence of sumatriptan. The mRNA expression of 5-HT_1B, 5-HT_1D, and 5-HT₇ receptors was not observed in the rat anococcygeus muscle. The results indicate that sumatriptan causes relaxation of the precontracted rat anococcygeus muscle by a calcium-antagonistic activity.

Preservation of the Endothelium in Cardiovascular Surgery – Some Practical Suggestions – A review

The most vulnerable structure in vascular tissue is the endothelium, which plays a critical role in the maintenance of normal vessel wall structure and function. Endothelial cells produce a variety of biologically active substances which control vascular permeability, vessel tone, coagulation, Ž brinolysis and in ammatory responses. Some of these substances, such as the proteins of cellular junctions or adhesion molecules, are integral parts of the cell structure whereas others, such as nitric oxide (NO), prostacyclin, chemokines, or factors involved in coagulation and Ž brinolysis, are produced and then released by endothelial cells either luminally or abluminally (1). The endothelium is antithrombogenic when well preserved, and yet promotes platelet aggregation and coagulation if injured. Vasospasm, occlusive intimal hyperplasia and accelerated atherosclerosis can also all occur as a result of bad endothelial preservation (2). The persons who are faced most directly with the problems of endothelial preservation in cardiovascular surgery are the surgeons transplanting hearts and lungs; if the endothelium is badly preserved in the donor organ, the blood cells of the recipient may not be able to pass through the capillaries due to endothelial swelling, and multiple infarctions and death of the recipient may occur. In most published data on heart and lung transplantation, the early mortality due to “primary organ failure” is in the range of 10–25%, attesting to the signiŽ cance of the problem. Coronary surgeons are also transplantation surgeons: they harvest blood vessel segments, veins or arteries, and transplant them into the coronary circulation. Although this is autotransplantation, it is still transplantation, and the complex and vulnerable vascular tissue they transplant should be preserved using the same principles as those used by transplant surgeons in general. During prolonged cardiopulmonary bypass (CPB), the lungs may suffer ischemic injury and the use of cold crystalloid cardioplegia delivered under high pressure into the coronary arteries may elicit endothelial injury, initiating vasospasm and accelerated atherosclerosis. This review article will give some practical suggestions on how to minimize these problems. Once every minute the entire blood volume passes through the pulmonary artery, which could be compared with a big vascular tree, branching out to meet the air in the alveolocapillary membrane, making possible the vital gas exchange of the blood. During CPB blood still  ows in the pulmonary circulation, coming from the bronchial arteries, but it is important to keep in mind in these days of “warm heart surgery” (3), that the bronchial circulation alone cannot supply the lungs with enough blood during normothermia to prevent ischemic lung injury (4, 5). If the bypass time for unexpected reasons needs to be prolonged, cooling or pulmonary artery perfusion should be initiated. The young cardiac surgeon should never forget that the golden standard temperature used for many years during CPB was 28°C, and that many senior surgeons even today cool their patients routinely to at least 32°C during CPB. In pigs, at least 25% of the cardiac output must be directed through the pulmonary artery at normothermia to prevent ischemic lung injury (5). If the cardiac output can be directed through the pulmonary artery, total extracorporeal membrane oxygenation (veno-right ventricular ECMO) supplying a patient’s whole need for gas exchange, can be run for several weeks without causing lung injury (6–8). However, total veno-arterial ECMO, bypassing the pulmonary artery circulation, cannot be run for more than a few hours before ischemic injury of the lung vasculature occurs, manifesting itself after weaning from bypass as increased pulmonary vascular resistance or intrapulmonary bleeding (4, 5). In lung transplantation, preservation of the donor lungs has until recently been obtained by cold  ush perfusion of the pulmonary artery with Euro-Collins solution, a high-potassium kidney preservation solution developed in the 1960s. For preservation of vascular tissue, Euro-Collins solution is inferior to Perfadex (9), a low-potassium dextran (LPD) solution, also developed in the 1960s as a kidney preservation solution. Perfadex gives excellent lung (10) and vascular (9, 11) preservation for 24 h. By switching over from EuroCollins to Perfadex in clinical lung transplantation,

Different distribution of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat hippocampal neurons.

The distribution patterns of nifedipine- and omega-conotoxin GVIA-sensitive Ca2+ channels in rat primary cultured hippocampal neurons were investigated with a confocal laser-scanning microscope. Cells were loaded with the calcium indicator dye Oregon Green/AM, and the responses to high potassium (90 mM) solution with and without the existence of L- and N-type Ca2+ channel blockers were imaged. In general, extracellular application of high [K+] solution induced a [Ca2+]o-dependent increase of [Ca2+]i in both somata and dendritic processes. The increase was reduced by a N-type channel blocker, omega-conotoxin GVIA, and the reduction was greater in dendritic processes than in somata and proximal dendrites. In contrast, the reduction induced by the L-type calcium channel blocker, nifedipine, was observed evenly all over the neurons. The results demonstrated the heterogeneous distribution of nifedipine- and omega-conotoxin GVIA-sensitive calcium channels in cultured hippocampal neurons.

Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus

ATP and adenosine are well-known neuroactive compounds. Other nucleotides and nucleosides may also be involved in different brain functions. This paper reports on extracellular concentrations of nucleobases and nucleosides (uracil, hypoxanthine, xanthine, uridine, 2′-deoxycytidine, 2′-deoxyuridine, inosine, guanosine, thymidine, adenosine) in response to sustained depolarisation, using in vivo brain microdialysis technique in the rat thalamus. High-potassium solution, the glutamate agonist kainate, and the Na +/K + ATPase blocker ouabain were applied in the perfusate of microdialysis probes and induced release of various purine and pyrimidine nucleosides. All three types of depolarisation increased the level of hypoxanthine, uridine, inosine, guanosine and adenosine. The levels of measured deoxynucleosides (2′-deoxycytidine, 2′-deoxyuridine and thymidine) decreased or did not change, depending on the type of depolarisation. Kainate-induced changes were TTX insensitive, and ouabain-induced changes for inosine, guanosine, 2′-deoxycytidine and 2′-deoxyuridine were TTX sensitive. In contrast, TTX application without depolarisation decreased the extracellular concentrations of hypoxanthine, uridine, inosine, guanosine and adenosine. Our data suggest that various nucleosides may be released from cells exposed to excessive activity and, thus, support several different lines of research concerning the regulatory roles of nucleosides.

Tumor necrosis factor-α augments contraction and cytosolic Ca 2+ sensitivity through phospholipase A 2 in bovine tracheal smooth muscle

To elucidate the effects of tumor necrosis factor-α (TNF-α) on tracheal smooth muscle contraction, we simultaneously measured isometric tension and intracellular Ca 2+ ([Ca 2+] i) in fura 2-loaded muscle strips. Smooth muscle force generation was evaluated in a high potassium (K +; 20.0–80.0 mM) solution and with acetylcholine (3 nM–10 μM ). TNF-α (1–100 ng/ml) did not directly contract muscle strips. The contractile response to acetylcholine was enhanced after application of 10 ng/ml of TNF-α for 30 min but not the response of [Ca 2+] i. The contractile response and the response of [Ca 2+] i to a high K + solution were not altered after application of TNF-α. The [Ca 2+] i–tension curve indicated that TNF-α enhanced the responsiveness of tracheal smooth muscle through the acetylcholine-mediated Ca 2+ sensitivity of intracellular contractile elements. The augmentation of the acetylcholine concentration–response curves for muscle tension in the presence of TNF-α (10 ng/ml) was inhibited in part after application of manoalide, a phospholipase A 2 inhibitor. We conclude that a low concentration of TNF-α enhances smooth muscle responsiveness to acetylcholine by agonist-mediated Ca 2+ sensitivity facilitated by phospholipase A 2.

Reactive oxygen species-induced aortic vasoconstriction and deterioration of functional integrity.

Oxygen derived free radicals and other reactive oxygen species (ROS) are involved in a variety of disease states, which can have cardiac and vascular implications. The present study was performed to investigate the mechanism of ROS-induced vasoconstriction and the influence of ROS on the functional integrity of isolated rat thoracic aorta. ROS were generated by means of electrolysis (30 mA, during 0.5, 1, 2 or 3 min) of the organ bath fluid. ROS induced a transient (approximately 60 min) vasoconstriction and the maximally induced contraction was dependent on the duration of electrolysis. Dimethyl sulfoxide (DMSO) diminished the ROS-induced vasoconstriction almost completely, indicating a major influence of hydroxyl radicals on contractility. The dual cyclooxygenase/lipoxygenase inhibitor, meclofenamate, completely prevented the ROS-induced vasoconstriction. The phospholipase A2 (PLA2) inhibitor, oleyloxyethyl phosphorylcholine, was able to reduce the vasoconstriction elicited by ROS by approximately 70%. Conversely, the specific cytoplasmic PLA2 inhibitor arachidonyl trifluoromethylketone proved ineffective in this respect. By using the specific mitogen-activated protein kinase (MAPkinase) kinase inhibitor PD98059, it was shown that the activation of extracellular-regulated kinase (ERK) MAPkinase contributes to the ROS-induced vasoconstriction. The effects of ROS on the functional integrity of the aortae were investigated, in particular with respect to receptor (alpha1-adrenoceptor) and non-receptor-mediated contractile responses (high potassium solution). In addition, both the endothelium dependent (methacholine) and endothelium independent (sodium nitroprusside) vasorelaxation were investigated before and after ROS exposure. Electrolysis periods of 0.5 and 1 min induced a modest leftward shift of the concentration response curves for the alpha1-adrenoceptor agonist methoxamine. Longer electrolysis periods of 2 and 3 min additionally decreased the maximal response to (alpha1-adrenoceptor stimulation. Methacholine-induced vasorelaxation proved diminished in aortae subjected to electrolysis (0.5, 1, 2 and 3 min), whereas relaxation to sodium nitroprusside was nearly complete in all groups. KCl-induced contractions proved attenuated only after longer electrolysis periods of 2 and 3 min. This ROS-induced deterioration of functional integrity was almost completely prevented by 0.6% DMSO. From these results we may conclude that ROS induce an eicosanoid and ERK MAPkinase-mediated vasoconstriction in isolated rat thoracic aorta. In addition, exposure to ROS leads to a deterioration of functional integrity characterized by endothelial dysfunction and decreased contractile function.

Gating and Flickery Block Differentially Affected by Rubidium in Homomeric KCNQ1 and Heteromeric KCNQ1/KCNE1 Potassium Channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast “flickery/block” process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.

Attachment of growth cones on substrate observed by multi-mode light microscopy.

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.

Distribution of nifedipine- and ω-conotoxin GVIA-sensitive Ca 2+ channels in cultured rat neocortical neurons

L- and N-type voltage-dependent calcium channels are widely distributed in neurons of the CNS. To investigate their subcellular distributions on CNS neurons, intracellular calcium concentration ([Ca 2+] i) increase in response to high potassium ([K +]) solution was detected in primary cultured rat neocortical neurons using the calcium indicator dye Oregon Green with a confocal laser scanning microscope. Extracellular application of 90 mM [K +] solution induced fluorescence increase in a manner dependent on extracellular [Ca 2+]. The increase was partially blocked by 10 μM nifedipine, and the reduction was higher in cell bodies compared to dendritic processes. In contrast, ω-conotoxin GVIA reduced the 90 mM [K +] induced fluorescence increase more in the dendritic processes. The results demonstrated the heterogeneous distribution of nifedipine- and ω-conotoxin GVIA-sensitive calcium channels, which may suggest a functional difference in nifedipine- and ω-conotoxin GVIA-sensitive channels in cultured neocortical neurons.

Regulation of vascular tone by endothelium-derived hyperpolarizing factor.

1. Endothelium-derived hyperpolarizing factor (EDHF) mediates the nitric oxide (NO)-independent component of the relaxation in rat mesenteric arteries. The relationship between hyperpolarization and vascular tone was studied by simultaneous recording of membrane potential with intracellular microelectrodes and tension in ring segments of rat mesenteric arteries. 2. By depolarizing arteries with high potassium solutions, it was determined that the threshold for contraction is approximately -46 mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. Thus, 1 mV depolarization resulted in an approximate 4% increase in tone. This relationship was not altered in spontaneously hypertensive rats. 3. Noradrenaline (0.3 mumol/L) caused contraction and depolarized arteries by 13 mV. Acetylcholine caused endothelium-dependent relaxation and hyperpolarization up to 14 mV. In the presence of N omega-nitro-L-arginine, the EDHF-mediated relaxation was correlated to hyperpolarization. A hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone. 4. At concentrations (10 mumol/L) causing total relaxation, the maximum hyperpolarization induced by NO was only 7.6 mV. 5. A maximum relaxation of 88% was observed with pinacidil (3 mumol/L), despite a 25 mV hyperpolarization. Relaxations to NO and pinacidil were not correlated with hyperpolarization. At similar levels of hyperpolarization, NO and pinacidil elicited more relaxation than EDHF. 6. These studies show that vascular tone is very sensitive to membrane potential change in the range between -46 and -20 mV in the rat mesenteric artery. The relaxation response to EDHF, unlike that to NO and pinacidil, can be accounted for solely by its effect on the membrane potential.

Does PGE 1 attenuate potassium-induced vasoconstriction in initial pulmonary artery flush on lung preservation?

The standard clinical protocol for lung transplantation employs cold single pulmonary artery flush with Euro-Collins solution or the University of Wisconsin solution. Prostaglandin E 1 (PGE 1) is usually given by direct injection into the pulmonary artery to reduce pulmonary vasoconstriction caused by these intracellular, high-potassium solutions, however, the efficacy of PGE 1 on lung preservation remains controversial. In this study we demonstrated that vasodilator effects of PGE 1 were markedly reduced under a high-potassium condition, and that potassium-induced pulmonary vasoconstriction were inhibited by calcium channel blocker nifedipine. There are three therapeutic options in the cold single pulmonary artery flush for optimal lung transplantation, including the use of a higher dose of PGE 1, use of the calcium channel blocker instead of PGE 1, or the use of the extracellular, low-potassium solution such as low-potassium dextran solution for initial pulmonary artery flush before the lung harvest.

An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture.

1. A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation. 2. The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothelial cells from porcine coronary arteries. 3. Calcium imaging was performed using the calcium-sensitive dye fura-2. A wound or a delivery of bradykinin to two to three cells on growing clusters of approximately 300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 micrometer. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid. 4. The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic decrease in the number of connexin 40 gap junctions in the confluent cultures. 5. The regenerative propagation of the wave was blocked by inhibitors of calcium-induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high-potassium solution. 6. We conclude that regenerative intercellular calcium waves exist in cultured islets but not in confluent cultures of endothelial cells. An increase in [Ca2+]i is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully regenerated propagation.

Vasorelaxant effects induced by the antiangiogenic drug linomide in aortic and saphenous vein preparations of the rabbit

1. Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carb oxa mide) inhibits vascular proliferation and has been proposed as an antiangiogenic drug. We have investigated the vascular effect of linomide in rabbit aortic and saphenous vein ring preparations and in rat cultured vascular smooth muscle cells (VSMCs). 2. Linomide (25-300 micrograms ml-1) did not alter the basal tone of the preparations. The drug induced a concentration-dependent relaxant effect in aortic rings with endothelium, preconstricted by noradrenaline (NA), 5-hydroxytryptamine (5-HT) and by the thromboxane mimetic U46619. 3. The degree of relaxation induced by linomide was significantly reduced by exposure to the cyclooxygenase inhibitors indomethacin (3 microM) and acetylsalicylic acid (500 microM), and was not influenced by pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (100 microM) in aortic rings with endothelium, preconstricted with NA. 4. Endothelium removal significantly reduced the relaxant response to linomide in aortic ring preparations. 5. A concentration-dependent relaxant response was observed also in rabbit saphenous vein preparations deprived of endothelium and preconstricted either by NA or U46619. The degree of relaxation obtained in a high potassium solution was consistently smaller than that observed in NA-pretreated venous preparations. 6. The vasorelaxant effect of linomide was consistently blunted by the adenylate cyclase inhibitor SQ 22536 (50 microM), both in intact aortic rings and in those deprived of endothelium. 7. In rat cultured vascular smooth muscle cells, linomide (100-200 micrograms ml-1) induced a significant increase in cyclic AMP levels, which was blocked by exposure to 50 microM SQ 22536. 8. In endothelium-deprived aortic ring preparations, the linomide-induced relaxant effect was greatly reduced in high potassium medium (KCl = 25 mM). Pretreatment with the ATP potassium channel inhibitor glibenclamide (3 microM) significantly reduced the linomide-induced relaxation. 9. The results show that linomide possesses a vasorelaxant effect which is attributable to both endothelium-dependent and -independent properties. While the former component of the drug's activity is apparently due to the release of a prostanoid from endothelial cells, the endothelium-independent mechanism involved in linomide relaxation is linked to cyclic AMP accumulation and to ATP-sensitive potassium channel activation in VSMCs.

Interactions between neuropeptide Y and the adenylate cyclase pathway in rat mesenteric small arteries: role of membrane potential.

1. Simultaneous measurements of membrane potential and tension were performed to investigate the intracellular mechanisms of neuropeptide Y (NPY) in rat mesenteric small arteries. 2. NPY (0.1 microM) depolarized arterial smooth muscle cells from -55 to -47 mV and increased wall tension by 0.22 N m-1, representing 11% of the contraction elicited by a high-potassium solution. Isoprenaline (1 microM) and acetylcholine (1 microM) evoked hyperpolarizations of 11 and 17 mV, respectively. NPY inhibited the isoprenaline-induced effects on membrane potential without affecting those of acetylcholine. 3. Forskolin evoked sustained concentration-dependent hyperpolarizations of small mesenteric arteries. NPY (0.1 microM) inhibited the responses to 1 microM forskolin, but did not alter the stable hyperpolarization elicited by the specific activator of protein kinase A (PKA) SP-5,6-DCl-cBIMPS (0.1 mM). Forskolin increased the cyclic AMP (cAMP) content of the arteries 21-fold, and NPY inhibited the forskolin-evoked increase in cAMP levels by 91%. 4. The hyperpolarization produced by 1 microM forskolin was not affected by either charybdotoxin (0.1 microM) or 4-aminopyridine (0.5 mM), but glibenclamide (5 microM) inhibited the hyperpolarization by 70%. Glibenclamide also inhibited the hyperpolarization evoked by SP-5,6-DCl-cBIMPS by 59%. 5. Neither depolarization nor contraction caused by NPY were significantly affected by either glibenclamide (5 microM) or nifedipine (1 microM), but they were reduced by gadolinium (10 microM). However, the blocking effect of NPY on forskolin-elicited hyperpolarization was not affected by gadolinium. 6. Charybdotoxin (0.1 microM) and 4-aminopyridine (0.5 mM) strongly enhanced the depolarization and contraction caused by NPY (0.1 microM), and nifedipine (1 microM) prevented the enhanced responses to NPY in the presence of charybdotoxin. 7. These findings suggest that NPY acts through at least two different intracellular mechanisms in mesenteric small arteries: a depolarization of arterial smooth muscle which is probably due to activation of non-selective cation channels, and a marked inhibition of adenylate cyclase activity, which in turn inhibits the hyperpolarization produced by cAMP accumulation in these arteries.

The Effect of Anoxia and Glucose-Free Solutions on the Contractile Response of Guinea-Pig Detrusor Strips to Intrinsic Nerve Stimulation and the Application of Excitatory Agonists

Purpose To study the effects of anoxia and substrate depletion, both separately and combined, on the contractile responses of guinea-pig detrusor smooth muscle strips to activation of intrinsic nerves, application of agonists and depolarization with high potassium solution. Materials and Methods Bladders were opened and the urothelium removed. Strips of detrusor were dissected and mounted for tension recording in small organ baths superfused with warmed solutions of known composition. Results Anoxia caused a small initial reduction of the contractile responses which then remained constant for several hours. Glucose-free solutions resulted in a slow progressive decline in the responses to field stimulation, carbachol and high potassium solution, with substantial responses still present after five hours. The response to ATP, however, was unaffected. Removal of oxygen and glucose, mimicking ischaemia, caused abolition of all responses within an hour. After reintroduction of normal conditions responses reached their maximal extent of recovery within an hour. Recovery was almost complete for responses to ATP and carbachol, but less so far high K sup + solutions. Very little recovery to stimulation of intrinsic nerves was seen. Conclusions The results demonstrate that phasic contractions can be fuelled almost fully by oxidative phosphorylation or by anaerobic glycolysis, but that in conditions mimicking ischaemia the intrinsic nerves are more susceptible to ischaemic damage than the detrusor smooth muscle.

Role of eicosanoids in alteration of membrane electrical properties in isolated mesenteric arteries of salt-loaded, Dahl salt-sensitive rats.

1. The role of eicosanoids in altered membrane electrical properties of Dahl salt-sensitive (DS) rats was investigated, by use of conventional microelectrodes technique, in isolated superior mesenteric arteries of DS rats and Dahl salt-resistant (DR) rats fed either a high or low salt diet. 2. The membrane was significantly depolarized in salt-loaded DS rats compared with the other three groups. In addition, the arteries of salt-loaded DS rats exhibited spontaneous electrical activity. 3. Spontaneous electrical activity in salt-loaded DS rats was inhibited by the following: indomethacin, a cyclo-oxygenase inhibitor; ONO-3708, a prostaglandin H2/thromboxane A2 receptor antagonist; OKY-046, a thromboxane A2 synthase inhibitor; nicardipine, a Ca(2+)-channel antagonist and by Ca(2+)-free solution. In addition, spontaneous electrical activity was enhanced by a thromboxane A2 analogue and by prostaglandin H2. Spontaneous electrical activity was unaffected by phentolamine, atropine and tetrodotoxin. 4. Membrane potential in arteries of salt-loaded DS rats was not affected by either indomethacin or ONO-3708. 5. Spontaneous contraction, sensitive to indomethacin, was present, and contractile sensitivity to high potassium solution was enhanced in arteries of salt-loaded DS rats. 6. These findings suggest that eicosanoid action, together with membrane depolarization, may lead to the activation of voltage-dependent Ca(2+)-channels, thereby causing spontaneous electrical activity in mesenteric arteries of salt-loaded DS rats. In addition, tension data suggest that these changes in membrane properties are related to enhanced contractile activities in salt-loaded DS rats. Mechanisms of depolarization remain to be determined.

In vivo elevation of extracellular potassium in the rat amygdala increases extracellular glutamate and aspartate and damages neurons

It is well known that high potassium (K +) solutions introduced by microdialysis into normal brain increase the extracellular concentration of the excitatory amino acid glutamate, and in vitro studies suggest that a high exogenously applied glutamate concentration can produce excitotoxic neuronal death. However, only recently were in vivo studies undertaken to determine whether high-K + exposure damages neurons. We implanted microdialysis probes into rat amygdalae bilaterally, and after a 2-h baseline period exposed one side to a modified Krebs–Ringer–bicarbonate solution containing 100 mmol/l KCl for 30, 50 and 70 min, followed by a 2-h recovery period, and 70 min and 3 h without a recovery period. Of 100.9±2.0 mmol/l KCl, 12.0±1.0% was extracted by amygdalar tissue in vivo. Elevation of the extracellular K + concentration in the amygdala for 70 min or longer without a recovery period produced extensive neuronal damage and edematous-appearing neuropil in the tissue dialysed, as well as loss of normal neurons. Histological evidence of edema subsided in the groups with a 2-h recovery period. Although the number of damaged neurons was not significantly higher in the group with a 70 min high-K + exposure and 2-h recovery period, the number of normal neurons was reduced, suggesting cell loss. During 70-min high-K + exposure, the extracellular glutamate concentration increased to 242–377% of baseline during the first 60 min, and extracellular aspartate rose to 162–213% during the first 50 min; extracellular taurine rose even higher, to 316–567% of baseline, and glutamine fell to 14–27% of baseline. Extracellular serine was decreased at 20, 50 and 70 min of high-K + exposure; extracellular glycine was unchanged. The elevated extracellular glutamate and aspartate concentrations suggest that exposure of the amygdala to high extracellular K + may produce cell death through an excitotoxic process, and point the way to future studies to define the specific mechanisms involved.

Effects of Thapsigargin and Cyclopiazonic Acid on Intracellular Calcium Activity in Newborn Rat Cardiomyocytes During Their Development in Primary Culture

The effects of specific inhibitors of sarcoplasmic reticulum (SR) calcium ATPase, thapsigargin (TG), and cyclopiazonic acid (CPA) were investigated on the resting and transient levels of intracellular free calcium concentrations recorded in Indo-1-loaded ventricular myocytes of newborn rat heart in primary culture. The calcium transients were induced by caffeine (10 mM) or high potassium (100 mM) solutions. In 2 day- as in 7-day-old cultured cells, the calcium transients induced by 10 mM caffeine were blocked dose dependently by TG and CPA. The dose-response curves suggest that TG was more efficient than CPA and that both drugs were more efficient in 7-day- than in 2-day-old cells. The calcium transients induced by 100 mM K+ were also strongly inhibited by these agents. The lack of effect on sarcolemmal calcium currents, as shown by whole-cell patch-clamp experiments, suggests that these drugs affect only SR function. In cells exhibiting spontaneous activity, the associated calcium transients were not affected by TG or CPA at the beginning of the culture (2-day-old cells) but were fully blocked at the end (7-day-old cells). These results confirm that TG and CPA specifically inhibit the cardiac SR Ca2+ pump without affecting the sarcolemmal calcium current. Their blocking effect of the calcium transients as a function of the developmental stage of neonatal cardiac cells in culture suggests an increasing role of the SR in the regulation of intracellular calcium. This argues for developmental changes of the SR through the differentiation and maturation of newborn cardiomyocytes at the early stage of the postnatal life, leading to a predominant role of the SR in excitation-contraction coupling mechanisms in adult cells.

Effect of a brief stretch on time course of shortening after a quick release in guinea pig tenea coli.

To examine the origin of velocity deceleration in the shortening of smooth muscle, guinea pig tenea coli which contracts isometrically in a high-potassium solution was released quickly (1st release), restretched to the original length, kept at this length for 0.06-0.2s and quickly released again (2nd release). The muscle shortened isotonically under the same very-light load after the 1st and 2nd releases. The length change of the muscle during shortening was examined using a video camera. The rapid shortening phase, seen after the 1st release, which followed the instantaneous, elastic recoil and lasted for about 0.3s, disappeared after the 2nd release. This effect was prominent when the stretch duration was short. The time course of the slow phase of shortening subsequent to the rapid one was not affected by stretching. These results suggest that the rapid shortening phase is due largely to adjust of the cross-bridges between the thick and thin filaments to the change from an isometric to an isotonic contraction, while the slow phase reflects a steady turnover in the cross-bridges. The velocity of the slow shortening phase reached a maximum before the isometric force peaked and decreased as the activation level was lowered by reducing the external Ca2+ concentration from 2.5 to 0.5 mM. This suggests that the steady turnover rate of the cross-bridges depends on the phase of contraction and level of activation. The cause of the gradual velocity decrease in the slow phase of shortening was discussed.

Effect of verapamil on morphological differentiation and low-threshold calcium current of murine neuroblastoma cells

Changes in electrophysiological and morphological characteristics of N1E-115 murine neuroblastoma cells induced by their incubation in a medium containing verapamil, a blocker of high-threshold calcium channels, and/or increased concentrations of KCl were investigated. On the 3th day of cultivation with 1.0 µM verapamil, the cells showed morphological differentiation and increased density of low-threshold calcium current. Incubation of cells in a high-potassium solution (25 mM) produced inhibition of morphological differentiation and reduction of the density of this current. Morphological and electrophysiological parameters of cells cultivated in a high-potassium medium with verapamil added were close to those of control cells. Verapamil taken at a lower concentration (0.01 µM) did not affect the characteristics of calcium current and morphological differentiation of the cells. Higher concentrations of verapamil (100 µM) substantially suppressed viability of cells (a decrease in cells' projective area, retraction of cell processes, and sharp reduction of calcium current density were observed). The results are consistent with suppositions about functional significance of intracellular calcium level for cell differentiation.