High Potassium Solution Research Articles

Effect of short‐term organoid culture on the pharmaco‐mechanical properties of rat extra‐ and intrapulmonary arteries

1 Organoid cultured explants from differentiated tissues have gained renewed interest in the undertaking of physiological and pharmacological studies. In the work herein, we examined the pharmaco-mechanical properties of an in vitro model consisting of organoid cultured rings derived from rat extra- and intrapulmonary arteries, over a period of 4 days in culture. 2 Mechanical changes were quantified using isometric tension measurements on both fresh and cultured pulmonary arterial tissues, with experiments performed in the presence or absence of 10% foetal calf serum. Conventional histochemical and immunofluorescent stainings were also performed to assess tissue structure integrity and apoptosis. 3 The explants developed spontaneous rhythmic contractions (SRC) in approximately half of the vessels. SRC amplitude and time course were modified by conditions and agents acting on membrane potential (high-potassium solutions--levcromakalim, a potassium channel opener), while nitrendipine, an L-type calcium channel blocker, suppressed SRC. 4 Cultured explants also developed a hyper-reactivity to high potassium challenges (10-40 mM). Whereas contraction to serotonin (5-HT) was enhanced in intrapulmonary arteries, contraction to endothelin-1 remained unchanged after 4 days of culture. Serum did not alter contractile properties during the culture period. 5 Endothelial-dependent relaxation was maintained in response to A23187 500 microM, but was abolished in response to 10 microM carbamylcholine. 6 Histological and immuno-histological analyses revealed the absence of hypertrophied vascular wall or apoptosis. 7 In conclusion, the contractile phenotype as well as tissue structure integrity of organoid explants remain essentially intact during short-term culture, making this model suitable for pharmaco-genomic studies.

Spontaneous Thermal Motion of the GABAA Receptor M2 Channel-lining Segments

The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

Evidence that 17alpha-estradiol is biologically active in the uterine tissue: Antiuterotonic and antiuterotrophic action

Background17alpha-Estradiol has been considered as the hormonally inactive isomer of 17beta-estradiol. Recently, nongenomic (smooth muscle relaxation) and genomic (light estrogenic activity) effects of 17alpha-estradiol have been reported, but no reports have yet determined its possible antiestrogenic activity. Therefore, this study investigated: the nongenomic action of 17alpha-estradiol on uterine contractile activity and its potential agonist-antagonist activity on uterine growth.MethodsUterine rings from rats were isometrically recorded. Different concentrations (0.2–200 microM) of 17alpha-estradiol were tested on spontaneous contraction and equimolarly compared with 17beta-estradiol. To examine the mechanism of 17alpha-estradiol action, its effect was studied in presence of beta2-antagonist (propranolol), antiestrogens (tamoxifen and ICI 182,780) or inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D). Moreover, contractions induced by high potassium (KCl) solution or calcium in depolarized tissues by KCl-calcium free solution were exposed to 17alpha-estradiol. Collaterally, we performed an uterotrophic assay in adult ovariectomized rats measuring the uterine wet weight. The administration for three days of 0.3 microM/day/Kg 17beta-estradiol was equimolarly compared with the response produced by 17alpha-estradiol. Antiuterotrophic activity was assayed by administration of 0.3 microM/day/Kg 17beta-estradiol and various doses ratios (1:1, 1:3, 1:5, and 1:100) of 17alpha-estradiol.ResultsThe estradiol isomers elicited an immediate relaxation, concentration-dependent and reversible on spontaneous contraction. 17alpha-Estradiol presented lower potency than 17beta-estradiol although it did not antagonize 17beta-estradiol-induced relaxation. Relaxation to 17alpha-estradiol was not inhibited by propranolol, tamoxifen, ICI 182,780, cycloheximide or actinomycin D. The KCl contractions were also sensitive to 17alpha-estradiol-induced relaxation and calcium contractions in depolarized tissues were markedly prevented by 17alpha-estradiol, implying a reduction of extracellular calcium influx through voltage-operated calcium channels (VOCCs). Uterotrophic assay detected significant increase in uterine weight using 17alpha-estradiol, which was significantly minor as compared with 17beta-estradiol. 17alpha-Estradiol, at all doses ratios, significantly antagonized the hypertrophic response of 17beta-estradiol.Conclusion17alpha-Estradiol induces a relaxing effect, which may be independent of the classical estrogen receptor, nongenomic action, apparently mediated by inactivation of VOCCs. 17alpha-Estradiol is also a weak estrogen agonist (uterotrophic response); likewise, 17alpha-estradiol may act as an antiestrogen (antiuterotrophic response). The overall data document a nongenomic relaxing action and a novel antiestrogenic action of 17alpha-estradiol, which are relevant in estrogen-mediated uterine physiology.

Androgens Induce Relaxation of Contractile Activity in Pregnant Human Myometrium at Term: A Nongenomic Action on L-Type Calcium Channels1

It has long been accepted that progesterone regulates uterine contractile activity. However, little is known about the role of androgens in female physiology, and their importance and biological function on myometrial contractility so far have received limited attention. In this work, we examined the direct effect of androgens on the contractile activity of the isolated human myometrium. Myometrial biopsies were obtained, with consent, from pregnant women undergoing elective cesarean section at term. Each androgen tested (dehydroepiandrosterone, testosterone, 5alpha- and 5beta-dihydrotestosterone, androsterone, or androstanediol) caused a concentration-dependent inhibition of spontaneous contractile activity; a relaxing effect of these androgens was also observed on the contractions induced by high potassium (KCl) solution. Interestingly, nonpregnant myometrium was also sensitive to androgen-induced relaxation. 5beta-Dihydrotestosterone (5beta-DHT) was dramatically more potent than the other androgens in inducing myometrial relaxation in all preparations. Relaxation response to androgens had very rapid time courses and was affected by neither the specific antiandrogen (flutamide) nor inhibitors of protein synthesis (cycloheximide) and transcription (actinomycin D), implying that androgens act through a nongenomic mechanism. Importantly, 5beta-DHT significantly reduced the increase in intracellular calcium concentration associated with exposure to KCl in human myometrial smooth-muscle cells loaded with Fura-2-AM. The blockade of l-type calcium channels seems to be involved in the nongenomic relaxing action of androgens. These observations demonstrate that androgens may play a crucial role in maintaining pregnancy.

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to −80 °C at 1 °C/min and stored in liquid nitrogen for 7–36 days. Samples were thawed in a 37 °C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm 2 for 3–5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 ± 5% and a viability of 79 ± 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.

A secondary metabolite, 4,5-dibromopyrrole-2-carboxylic acid, from marine sponges of the genus Agelas alters cellular calcium signals

A secondary metabolite from sponges of the genus Agelas, 4,5-dibromopyrrole-2-carboxylic acid, which is well known as feeding deterrent, was investigated for effects on the cellular calcium homeostasis in PC12 cells. 4,5-Dibromopyrrole-2-carboxylic acid did not change intracellular calcium levels if applied alone without cell depolarization. During depolarization of the cellular membrane using high potassium solution, a dose dependent reduction of intracellular calcium elevation was revealed utilizing Fura II as calcium indicator. Significant reduction was seen at concentrations higher than 30 μM in a series of experiments, but in single experiments a concentration of 300 nM was still reversible effective. In the same concentration range, the onset of depolarization induced calcium elevations was significantly delayed by 4,5-dibromopyrrole-2-carboxylic acid. Dose dependent reduction and delay of depolarization evoked calcium elevations are probably due to a reduction of calcium entry via voltage operated calcium channels. One cellular mode of action of the feeding deterrent potential of 4,5-dibromopyrrole-2-carboxylic acid to fishes may be an interaction with the cellular calcium homeostasis of exposed cells.

Major role for tonic GABAA conductances in anesthetic suppression of intrinsic neuronal excitability.

Anesthetics appear to produce neurodepression by altering synaptic transmission and/or intrinsic neuronal excitability. Propofol, a widely used anesthetic, has proposed effects on many targets, ranging from sodium channels to GABA(A) inhibition. We examined effects of propofol on the intrinsic excitability of hippocampal CA1 neurons (primarily interneurons) recorded from adult rat brain slices. Propofol strongly depressed action potential production induced by DC injection, synaptic stimulation, or high-potassium solutions. Propofol-induced depression of intrinsic excitability was completely reversed by bicuculline and picrotoxin but was strychnine-insensitive, implicating GABA(A) but not glycine receptors. Propofol strongly enhanced inhibitory postsynaptic currents (IPSCs) and induced a tonic GABA(A)-mediated current. We pharmacologically differentiated tonic and phasic (synaptic) GABA(A)-mediated inhibition using the GABA(A) receptor antagonist SR95531 (gabazine). Gabazine (20 microM) completely blocked both evoked and spontaneous IPSCs but failed to block the propofol-induced depression of intrinsic excitability, implicating tonic, but not phasic, GABA(A) inhibition. Glutamatergic synaptic responses were not altered by propofol (< or =30 microM). Similar results were found in both interneurons and pyramidal cells and with the chemically unrelated anesthetic thiopental. These results suggest that suppression of CA1 neuron intrinsic excitability, by these anesthetics, is largely due to activation of tonic GABA(A) conductances; although other sites of action may play important roles in affecting synaptic transmission, which also can produce strong neurodepression. We propose that for some anesthetics, suppression of intrinsic excitability, mediated by tonic GABA(A) conductances, operates in conjunction with effects on synaptic transmission, mediated by other mechanisms, to depress hippocampal function during anesthesia.

Kiss-and-run quantal secretion in frog nerve-muscle synapse.

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.

Effects of biochemical changes to filling media during urodynamic testing in women with lower urinary tract symptoms

Detrusor muscle is sensitive, in vitro, to changes in the chemistry of its environment. However, the extent to which the intravesical environment influences bladder function is unclear. This study was designed to assess the impact of changes to the intravesical biochemical environment on urodynamic testing, compared with a normal saline control. Ninety-four women were studied with repeat fill urodynamics, using a normal saline control and test solutions (hyperosmolar, a solution intended to cause extracellular alkalosis and solutions designed to affect both intracellular and extracellular pH, and high potassium solution). There were no significant effects of any changes in filling solution. Urodynamic testing was unaffected by changes to intravesical chemistry that are known to affect detrusor cells in vitro. This suggests that the homoeostatic mechanisms of the bladder are able to maintain a stable microenvironment for detrusor cells despite changes to bladder contents.

Electric field effects in hyperexcitable neural tissue: a review.

Uniform electric fields applied to neural tissue can modulate neuronal excitability with a threshold value of about 1 mV mm(-1) in normal physiological conditions. However, electric fields could have a lower threshold in conditions where field sensitivity is enhanced, such as those simulating epilepsy. Uniform electrical fields were applied to hippocampal brain slices exposed to picrotoxin, high potassium or low calcium solutions. The results in the low calcium medium show that neuronal activity can be completely blocked in 10% of the 30 slices tested with a field amplitude of 1 mV mm(-1). These results suggest that the threshold for this effect is clearly smaller than 1 mV mm(-1). The hypothesis that the extracellular resistance could affect the sensitivity to the electrical fields was tested by measuring the effect of the osmolarity of the extracellular solution on the efficacy of the field. A 10% decrease in osmolarity resulted in a 56% decrease (n = 4) in the minimum field required for full suppression. A 14% in osmolarity produced an 81% increase in the minimum field required for full suppression. These results show that the extracellular volume can modulate the efficacy of the field and could lower the threshold field amplitudes to values lower than approximately 1 mmV mm(-1).

Maturation and Release of Interleukin-1β by Lipopolysaccharide-primed Mouse Schwann Cells Require the Stimulation of P2X7 Receptors

The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1beta maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1beta but did not release detectable amounts of pro or mature IL-1beta. ATP on its own had no effect on the synthesis of pro-IL-1beta, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1beta by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1beta-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1beta in IL-1beta, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1beta. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1beta, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1beta release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1beta by immune-challenged Schwann cells.

Potassium-induced Slow Motility is Partially Calcium-dependent in Isolated Outer Hair Cells

Low flow rate (0.6 μl/min) administration of high concentration potassium solutions (12.5, 25 and 37.5 mM) was tested for evoking slow-motility length changes in isolated, apical turn, guinea pig outer hair cells (OHCs) (length 65–80 μm; n=38). Control OHCs (n=16) showed a flow rate-dependent, reversible, longitudinal shortening of 0.5–3 μm during perfusion with normal saline. Potassium, an effective depolarizing agent for OHCs, induced a concentration-dependent cell shortening of 0.5–13 μm. These cell shape changes were reversible. The magnitude of shortening was significantly (p<0.01) decreased in a calcium-free incubation medium (n=8). The velocity of the shortening was 300 nm/s in the first 10 s after application of 37.5 mM K+ in a normal incubation medium and decreased to 100 nm/s during the next 10 s. Corresponding velocities in calcium-free solutions were 100 and 50 nm/s, respectively. K+-induced shortening velocities were not significantly different from control values after 30 s. It appears that K+-induced OHC shortening is sensitive to the calcium content of the incubation medium during the first 10 s. Higher flow rate (1.5 μl/min) administration of K+ makes the velocity and magnitude of slow motility of OHCs insensitive to the absence of calcium. These results highlight the fact that one of the critical technical points in fluid perfusion experiments with isolated OHCs is selecting a safe low flow rate of <0.6 μl/min. At this perfusion rate, K+-induced OHC shortening is composed of both calcium-sensitive and -insensitive components.

Effects of chloroquine on smooth muscle contracted with noradrenaline or high-potassium solutions in the rat thoracic aorta.

The effects of chloroquine on the smooth muscle of isolated rat aortic segments were investigated in preparations contracted with either noradrenaline or high-potassium. At rest, chloroquine (up to 10(-4) M) produced no mechanical response, while noradrenaline (10(-6) M) produced a sustained contraction. In the presence of 10(-4) M chloroquine, however, the amplitude of contractions produced by noradrenaline was attenuated by about 70%, with no alteration of the resting tension. In preparations contracted either with noradrenaline or with high-K solutions, chloroquine produced a concentration-dependent relaxation. The tension decreased below resting level as a result of the co-application of these stimulants. The relaxing actions of chloroquine were not altered by methylene blue (an inhibitor of guanylate cyclase), suggesting that the cyclic GMP-related mechanism was not involved. The ratio of the amplitude of chloroquine-induced relaxation was similar in contractions produced by different concentrations of potassium ions, suggesting that chloroquine did not cause relaxation as a result of membrane hyperpolarization. These results suggest that the inhibition of aortic smooth muscle contraction caused by chloroquine is different to that produced by endothelium-derived vasodilating factors. It is possible that the inhibition of aortic smooth muscle contraction by chloroquine involves modulation of the contractile systems and of their regulatory proteins.

Mechanisms of heterogeneous endothelial cytoplasmic calcium increases in venular microvessels.

Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration, [Ca(2+)](i). We investigated the mechanisms that modulate localized increases in [Ca(2+)](i) in individual endothelial cells of microvessels after exposure to ATP. [Ca(2+)](i) was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca(2+)](i) from a localized mechanical stimulus was also measured. The peak [Ca(2+)](i) after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 +/- 5 nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca(2+)](i) had the largest fractional reduction. Localized increases in [Ca(2+)](i) due to mechanical stimulus did not spread. The key mechanism regulating the heterogeneity in initial peak increase in [Ca(2+)](i) is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca(2+)](i) in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.

TNFalpha and IL-1beta exert no direct vasoactivity in human isolated resistance arteries.

The proinflammatory cytokines TNFalpha and IL-1beta are thought to play a role in the mechanism of septic vasodilatory shock. Most evidence indicate that the vascular effects these cytokines are mediated through increased expression of inducible nitric oxide synthase, whereas several animal studies report a more rapid vasorelaxant effect. We studied the direct vascular effects of TNFalpha and IL-1beta in human isolated resistance arteries (n=9) using a microvascular myograph. After precontraction with noradrenaline or a depolarising high potassium solution, TNFalpha and IL-1beta (up to 10(-8) M, alone or in combination) did not exert any relevant vasoactive effect. We conclude that, in contrast with some animal experiments, these cytokines exert no direct vasoactivity in human resistance arteries.

Effects of AMPA-receptor and voltage-sensitive sodium channel blockade on high potassium-induced glutamate release and neuronal death in vivo

High extracellular potassium induces spreading depression-like depolarizations and elevations of extracellular glutamate. Both occur in the penumbra of a focal ischemic infarct, and may be responsible for the spread of cell death from the infarct core to the penumbra. We have modeled this situation with microdialysis of an isotonic high-potassium solution into the normal rat amygdala for 70 min. This elevates extracellular glutamate up to 8-fold or more and produces irreversibly damaged, acidophilic neurons. NMDA-receptor blockade protects neurons and reduces the elevation of extracellular glutamate. Here we investigated the effects of sodium channel blockade with the voltage-sensitive sodium channel blocker tetrodotoxin and the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide disodium (NBQX disodium) on high potassium-induced neuronal death and extracellular glutamate elevations. The acidophilic neurons produced are necrotic by ultrastructural examination. Tetrodotoxin, at dialysate concentrations of 33, 330 and 3300 μM (only a small fraction is extracted by tissue), markedly reduced the elevations of glutamate in rat amygdala at nearly all time points during high-potassium perfusion, but it reduced tissue edema only at the highest concentration, and it was neuroprotective only if dialyzed prior to high-potassium microdialysis (at 330 μM concentration). Although both 250 μM (6.2% is extracted by tissue) and 500 μM NBQX reduced elevations of glutamate, neither was neuroprotective, and neuropil edema was not reduced by either concentration. Our results suggest that in vivo, sodium influx through voltage-sensitive sodium channels but not through ligand-gated AMPA receptor channels contributes to high potassium-induced neuronal necrosis.

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 °C. The cells were then cooled to −60 °C at controlled rates between 0.3 and 500 °C/min and stored below −180 °C. Samples were thawed in a 37 °C water bath and the cryoprotectant was removed by serial dilution at 22 °C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7–9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45–50% at cooling rates of 0.3, 1.0, and 10 ° C/min, but decreased to 20% at 50 °C/min and to <1% at 500 °C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 °C. Control, untreated confluent beads gave ∼73% of control uptake and negative controls (frozen without cryoprotectant) gave ∼4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 °C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 °C/min and to 85% at 0.3 °C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.

Novel receptors for ouabain: studies in adrenocortical cells and membranes.

Sodium-potassium pumps (Na pumps) are the only known plasma membrane receptors for cardiac glycosides. However, adrenocortical cells secrete an endogenous ouabain via an unknown mechanism that is subject to feedback inhibition via the cell surface. In addition, recent studies suggest that the induction of sustained hypertension by ouabain analogs in rats may be independent of Na pump inhibition. Accordingly, we used bovine adrenocortical cells and membranes to search for novel binding sites for ouabain. In high extracellular potassium solutions, the binding of ouabain to the Na pumps of cultured cells was suppressed, yet residual specific binding of (3)H-ouabain was observed. In high extracellular potassium, Scatchard analyses revealed a novel class of ouabain binding sites with high affinity (<50 nmol/L, < 2.5 x 10(5) sites/cell) that was distinct from the low-affinity Na pump sites (>1 micromol/L, 4.5 x 10(6) sites/cell). Analysis of the kinetics for the dissociation of (3)H-ouabain from intact cells revealed components whose t(0.5) values were 6.5 minutes, 3.3 hours, and 33 hours and associated with novel sites, Na pumps, and lysosomal recycling, respectively. Studies with isolated membranes under ligand conditions where the participation of Na pumps was minimized revealed specific ouabain binding to novel sites that was saturable, time-dependent, of high affinity (K(d) approximately 15 nmol/L), and of low density (apparent B(max)=0.23 pmol/mg, c.f., Na pumps=10.2 pmol/mg). Ouabain binding to the novel sites was stimulated by high concentrations of KCl but was not affected by aldosterone or cortisol up to 30 micromol/L. Novel sites were not detected in skeletal muscle or liver membranes. Photoaffinity studies followed by SDS-PAGE showed ouabain-protectable labeling of membrane polypeptides with apparent molecular weights of 143, 113, and 65 kDa. We conclude that adrenocortical cells express ouabain receptors that are distinct from Na pumps. These novel receptors may be involved in the regulation and/or secretion of endogenous ouabain.

Mechanisms of Heterogeneous Endothelial Cytoplasmic Calcium Increases in Venular Microvessels

Objective: Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration, [Ca2+]i. We investigated the mechanisms that modulate localized increases in [Ca2+]i in individual endothelial cells of microvessels after exposure to ATP. Methods: [Ca2+]i was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca2+]i from a localized mechanical stimulus was also measured. Results: The peak [Ca2+]i after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 ± 5 nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca2+]i had the largest fractional reduction. Localized increases in [Ca2+]i due to mechanical stimulus did not spread. Conclusion: The key mechanism regulating the heterogeneity in initial peak increase in [Ca2+]i is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca2+]i in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.

Development of pressurized retinal resistance-sized arteriolar preparation with special reference to acetylcholine-induced nitric-oxide-mediated vasodilatation.

We examined the responses of pressurized bovine retinal functional arterioles (97-185 microm in diameter and approximately 3 mm long) to vasoactive substances and the mode of action of acetylcholine (ACh) on the pressurized arterioles. The retinal arterioles were cannulated at both ends with glass micropipettes and perfused at a constant pressure of 60 mmHg. Vasoconstrictions of the retinal arterioles were induced by prostaglandin F(2 alpha) (PG F(2 alpha)), U46,619, noradrenaline (NA), and 5-hydroxytryptamine (5-HT) in a dose-dependent manner. The decreasing order of potency (pD(2) value) in the constrictive responses was as follows: 5-HT = U46,619 > NA > PG F(2 alpha). On the other hand, sodium nitroprusside (SNP), isocarbacyclin (a stable prostaglandin I(2) analog), ACh, and isoproterenol (ISP) caused dose-dependent vasodilatation in the pressurized retinal arterioles preconstricted with high-potassium solution (40 mM K+). The decreasing order of potency in the vasodilative responses was as follows: isocarbacyclin > SNP > ACh. The ACh-induced vasodilatation was suppressed significantly by pretreatment with N omega-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M). A treatment with L-arginine (10(-3) M) in the presence of 3 x 10(-5) M L-NAME reversed completely the L-NAME-induced reduction of the vasodilatation. These results suggest that ACh causes the production and release of endogenous nitric oxide or its related compounds, which results in vasodilatation of the pressurized bovine retinal functional arterioles.