Removal Of External Calcium Research Articles

The exit of mouse oocytes from meiotic M-phase requires an intact spindle during intracellular calcium release.

To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5'-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow re-formation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is microtubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.

Phosphatidate-induced arachidonic acid mobilization in mouse peritoneal macrophages.

Phosphatidate (PA) is synthesized by a variety of cells in response to physiological agonists. Addition of PA vesicles to [3H]arachidonic acid (AA)-labeled macrophages was found to induce the release of radiolabel in a dose- and time-dependent manner. This effect correlated with the uptake of PA by the macrophages and appeared to be attributable to PA itself and not to a PA metabolite. In parallel with AA release, PA induced a rapid increase in lysophosphatidylcholine in cells prelabeled with [14C]glycerol. Down-regulation of protein kinase C by long term exposure of the cells to phorbol myristate acetate or cell treatment with the protein kinase C inhibitor staurosporine did not affect the PA response. Also, removal of external calcium or cell treatment with the calmodulin antagonist trifluoperazine did not affect PA-induced AA release, while inhibiting the responses to zymosan, phorbol 12-myristate 13-acetate, and ionophore A23187. PA-induced AA release was not affected by intracellular calcium depletion by treatment with quin2/AM in the presence of EGTA. When assayed toward an AA-containing substrate, PA was able to enhance phospholipase A2 activity from cell homogenates in the absence of calcium. The dose dependence and magnitude of the PA effect correlated with those observed for PA-induced AA release in whole cells. Inclusion of ATP in the assay mixture did not affect the activity of the PA-stimulated phospholipase A2. These findings suggest a role for PA in the cascade of events leading to AA release in macrophages through Ca(2+)-independent stimulation of an AA-selective phospholipase A2.

HP 749 enhances calcium-independent release of [3H]norepinephrine from rat cortical slices and synaptosomes

Previous studies have shown that, at concentrations of 1 microM and 10 microM, HP 749 increased electrically-stimulated release of [3H]norepinephrine (NE) from rat cortical slices. These effects were Ca(2+)-dependent, indicating an effect on release from vesicular stores. At 100 microM, HP 749 had two effects. In addition to enhancing the Ca(2+)-dependent electrically-evoked release, it also induced a rise in the basal efflux (spontaneous release) of [3H]NE, which was observed in both cortical slices and synaptosomes. The spontaneous release effect was (1) not blocked by the reuptake inhibitor nomifensine, (2) not affected by removal of external calcium, (3) not blocked by vesicular depletion with reserpine, and (4) not inhibited by the sodium channel blocker tetrodotoxin (TTX). As would be expected, the spontaneous [3H]NE release induced by the cytoplasmic releaser tyramine and the sodium channel activator veratridine were blocked by nomifensine and TTX, respectively. Notably, however, the Ca(2+)-independent veratridine-induced release was completely blocked by 100 microM HP 749. The mechanism of spontaneous release of [3H]NE caused by 100 microM HP 749 is unresolved at present; however, the data are consistent with this release originating from a cytoplasmic source.

Captopril inhibits the agonist-induced increase of cytosolic free Ca2+ in glomerular mesangial cells

To evaluate the underlying mechanism of the putative renal protective effects of angiotensin converting enzyme (ACE) inhibitors, the modulatory action of captopril on the angiotensin II (Ang II) and platelet-derived growth factor (PDGF)-induced increase of cytosolic free calcium concentration ([Ca2+]i) was investigated in cultured glomerular mesangial cells (MC) from spontaneously hypertensive rats from the Münster strain (SHR) and normotensive Wistar-Kyoto rats (WKY). Resting [Ca2+]i was not affected by captopril in MC from either SHR or WKY. Captopril inhibited the Ang II-induced [Ca2+]i increase in MC from both SHR and WKY in a dose-dependent and time-dependent fashion. The preincubation of MC with 1 mumol/liter captopril for 40 minutes significantly reduced the Ang II-induced [Ca2+]i increase in SHR from 167 +/- 30 nmol/liter (N = 17) to 74 +/- 20 nmol/liter (N = 8, P < 0.05) and in WKY from 102 +/- 42 nmol/liter (N = 14) to 43 +/- 12 nmol/liter (N = 7, P < 0.05). After removal of external calcium there was no significant effect of captopril on the Ang II-induced [Ca2+]i increase. With the Mn2+ quenching technique, it was confirmed that captopril affects Ca2+ influx. Phospholipase C activity as estimated by diacylglycerol formation was not changed by captopril. The preincubation of MC with 1 mumol/liter captopril for 40 minutes significantly reduced the PDGF-induced [Ca2+]i increase in SHR from 166 +/-54 nmol/liter (N = 9) to 31 +/- 19 nmol/liter (N = 6, P < 0.01) and in WKY from 127 +/- 31 nmol/liter (N = 11) to 61 +/- 32 nmol/liter (N = 5, P < 0.05). Similarly captopril reduced the [Ca2+]i increase induced by endothelin and vasopressin. The results indicate that the actions of Ang II and PDGF on MC are modulated by captopril, probably resulting in the impairment of the calcium dependent contractile response of mesangial cells.

Alpha 1-adrenoceptors in rat dorsal raphe neurons: regulation of two potassium conductances.

1. alpha 1-Adrenoceptor activation caused two separate effects in rat dorsal raphe neurons: a depolarization and an increase in the duration of the after-hyperpolarization following the action potential. The depolarization often resulted in repetitive action potentials. The alpha 1-adrenoceptor antagonists prazosin and WB 4101 blocked the depolarization induced by phenylephrine. The concentration-response curve to phenylephrine was shifted to the right by WB 4101. 2. Under voltage clamp, alpha 1-adrenoceptor agonists caused an inward current at -60 mV, which often became smaller at negative potentials but rarely reversed polarity even at strongly negative potentials. Using whole-cell recording, the inward current reversed polarity at the equilibrium potential for potassium in the majority of cells. Intracellular Cs+ decreased or abolished the alpha 1-mediated inward current. The inward current was dependent on external calcium, but not on the degree of internal calcium buffering. Removal of external calcium or addition of MgCl2, CoCl2 or CdCl2 reduced or blocked the effects of alpha 1-adrenoceptor agonists. Barium and strontium supported and even augmented the inward current induced by alpha 1-adrenoceptor agonists, whereas nifedipine and omega-conous toxin had no effect. In contrast, internal dialysis with the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA) did not inhibit the inward current. 3. The alpha 1-induced depolarization was blocked (or occluded) by the inclusion of GTP-gamma-S (100 microM) in the recording pipette. The phorbol-ester 4-phorbol 12,13-dibutyrate (PDBu) had no action on the membrane potential and depressed the phenylephrine-induced depolarization. This depression was reversed by the non-selective protein kinase inhibitor staurosporin. 4. Phenylephrine and noradrenaline increased a late component of the after-hyperpolarization (late-AHP) that followed a single action potential. The alpha 1-sensitive late-AHP was blocked by apamine suggesting that it is a calcium-dependent potassium conductance. 5. Thapsigargin reduced the duration of the late-AHP and blocked the phenylephrine-mediated prolongation. Caffeine also augmented the late-AHP and ryanodine blocked the augmentation induced by caffeine. The augmentation induced by phenylephrine was not occluded by caffeine and was still present after the caffeine-induced augmentation was blocked by ryanodine. 6. In slices pretreated with manoalide the depolarization induced by alpha 1-agonists was not changed; however, the late-AHP was reduced in duration and the alpha 1-receptor-mediated augmentation of the late-AHP was decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes.

Connexin46 (cxn46) is a gap junctional protein that was cloned from a rat lens cDNA library. Expression of cxn46 in solitary Xenopus oocytes resulted in the development of a large time- and voltage-dependent current that was not observed in noninjected control oocytes or in oocytes injected with mRNA for cxn43 or cxn32. The cxn46-induced current activated at potentials positive to -20 mV. On repolarization to -40 mV, the current deactivated over a period of several seconds. Removal of external calcium caused a marked increase in the amplitude of the cxn46-induced current, shifted the steady-state activation curve to more negative potentials, and altered the kinetics of activation and deactivation. Increasing external calcium had the opposite effect. The ability of cxn46 to induce the formation of cell-to-cell channels was tested in the oocyte pair system. Oocyte pairs injected with cxn46 mRNA + antisense oligonucleotides for Xenopus cxn38 were strongly coupled. In contrast, oocyte pairs injected with antisense alone showed no coupling. The inactivation kinetics of the gap junctional channels resembled the deactivation kinetics of the cxn46-induced current in solitary oocytes.

Importance of Basal and Agonist-Stimulated Intracellular Calcium for Superoxide Anion Generation in Peritoneal Macrophages

The cellular signalling mechanisms initiated by platelet-activating factor (PAF) for stimulation of superoxide anion (O^–₂) generation in peritoneal macrophages were investigated by monitoring intracellular calcium ([Ca²⁺]_i) levels. In rat peritoneal macrophages, exogenous PAF elicited an increase in [Ca²⁺]_i, but there was no O^–₂ generation. PAF elicited similar increases in [Ca²⁺]_i in guinea pig macrophages, which were accompanied by O^-₂ generation. Removal of external calcium revealed that the major component of the PAF-induced increase in [Ca²⁺]_i, in both rat and guina pig peritoneal macrophages, was dependent on transmembrane Ca²⁺ flux. Pertussis toxin pretreatment had no effect on basal or PAF-induced increases in [Ca²⁺]_i. In guinea pig macrophages, removal of external calcium or pertussis toxin pretreatment reduced O^–₂ generation by approximately 50% and the combination of these pretreatments caused almost complete suppression of the response. Thus, additional second mesenger systems are activated in guinea pig macrophages following PAF receptor activation which appear to be essential for O^–₂ generation.

Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor.

In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.

K and Cl ion channels in the aqueous-facing membrane of the rat ciliary body epithelium

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 μM) but not by DIDS or DPC at 100 μM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2–50 μM) that inhibits the actions of calmodulin and by pBPB (10 μM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 μM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1–100 μM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 μM), tamoxifen (50 μM), and progesterone (100 μM) but not by others, e,g., forskolin (40 μM), dideoxyforskolin (40 μM), quinidine (100 μM) and cyclosporin A (10 μM).

Receptor-mediated effect of a synthetic thromboxane-analogue on cytosolic calcium in isolated proximal tubules

The aims of this study were to measure cytosolic calcium concentration -[Ca 2+]i- under resting conditions in isolated renal proximal tubules and to analyze the effect of U-46619 (stable analogue of thromboxane A 2/PGH 2 on [Ca 2+]i in a mammalian epithelium. Proximal tubules were dissected out from male New Zealand rabbits (2.5 to 3.0 kg). After isolation they were washed twice and resuspended in 2 ml phosphate buffer solution (PBS). Tubules were loaded with Quin 2-AM (25 μM) for 15 min. After washing with PBS to eliminate the excess of extracellular Quin 2, fluorescence was measured at 340 nm excitation and 490 emission, under resting conditions and after stimulation. U 46619 (from 10 nm to 10 mM) increased [Ca 2+]i in a concentration-dependent pattern. Exposure to an antagonist of the thromboxane receptor (S-145) blocked the response to U-46619. Removal of external calcium abolished the response to U-46619. Change of PBS for Ringer-choline blunted the response to thromboxane analogue. Our results indicate that U-46619 increases cytosolic calcium through a receptor-mediated mechanism that requires external calcium to operate. Blockade of the response in the absence of external sodium suggests that Na +/Ca 2+ exchanger participates in this response.

Effects of angiotensin II and arginine vasopressin on F-actin content of cultured mesangial cells.

The actin cytoskeleton of mesangial cells (MC) plays an important role in the contractile response to agonists as well as in the endocytosis of macromolecules. A quantitative study of the F-actin content of MC by the rhodamine-phalloidin binding assay was carried out. Angiotensin II (ANG II) (10(-6) M) significantly increased the F-actin content of MC by 30 min and at later time periods, with increases ranging from 31 to 46%. Arginine vasopressin (10(-8) M) produced a transient decrease of F-actin content of MC at 30 s but then significantly enhanced the F-actin content at later time periods. There was no change in total actin and protein content of MC at 30 min in the presence of either agent. Thus, the increase in F-actin is related to a shift in the G- to F-actin ratio and not to the synthesis of new F-actin. Because the incubation of MC with 1 (5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, did not attenuate the ANG II-induced increase in the F-actin content of MC, the shift does not appear to be mediated by the activation of protein kinase C. The removal of external calcium did not prevent the increase in F-actin. Dibutyryl cAMP (5 x 10(-4) M), a smooth muscle cell and MC relaxant, did not alter the F-actin content in MC, and 10(-5) M cytochalasin B significantly lowered F-actin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons.

1. The calcium influx pathway in large synaptic terminals of acutely isolated bipolar neurons from goldfish retina was characterized using Fura-2 measurements of intracellular calcium and patch-clamp recordings of whole-cell calcium current. 2. Depolarization of bipolar cells with high [K+]o resulted in a sustained, reversible increase in [Ca2+]i in both synaptic terminals and somata. Removal of external calcium abolished the response, as did the addition of 200 microM-cadmium to the bathing solution, indicating that the rise in [Ca2+]i was due to entry of external calcium. Dihydropyridine blockers of voltage-gated Ca2+ channels also blocked the influx, and the Ca2+ channel agonist Bay K 8644 potentiated influx, implicating voltage-activated, dihydropyridine-sensitive channels in the influx pathway. 3. Under voltage clamp, depolarization from a holding potential of -60 mV evoked a slowly inactivating inward current that began to activate at -50 to -40 mV and reached a maximal amplitude between -20 and -15 mV. This current was identified as a calcium current because it decreased when the extracellular calcium concentration was lowered, increased when barium was the charge carrier, and was blocked by 200 microM-external cadmium. The current was substantially blocked by 1 microM-nitrendipine and potentiated by 0.1 microM-Bay K 8644, as expected for L-type Ca2+ channels; it was unaffected by omega-conotoxin. No evidence for transient or rapidly inactivating Ca2+ current was found. 4. At a given level of potassium depolarization, both the amplitude and the speed of increase in [Ca2+]i were greater in synaptic terminals than in somata. For instance, depolarization by 32.6 mM-potassium caused an increase in intracellular calcium of 400 +/- 23 nM in terminals and 180 +/- 20 nM in somata (mean +/- S.E.M., n = 73 terminals, n = 30 somata), with maximal rates of change of 40 +/- 3 and 12 +/- 2 nM/s, respectively. 5. The contribution of terminal and somatic currents to the total whole-cell Ca2+ current was determined under voltage clamp by local application of calcium or of blocking agents. While there was no qualitative difference between currents in terminals and somata, synaptic terminals accounted for 64 +/- 3% (mean +/- S.E.M., n = 12) of the total whole-cell calcium current, and somata accounted for 39 +/- 2%. Thus, the density of Ca2+ current was higher in the terminal, accounting for the greater magnitude and speed of Ca2+ influx observed in terminals in Fura-2 experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

κ-opioid receptor stimulation increases intracellular free calcium in isolated rat ventricular myocytes

The effect of two specific ϰ-agonists, dynorphin A1–13 and U50, 488H, on intracellular free calcium [Ca] i in isolated rat ventricular myocytes was studied. A spectrofluorimetric method using fura 2 as calcium indicator was employed. It was found that both agonists increased [Ca] i dose-dependently. The effect was attenuated by Mr 2266, a κ-antagonist, indicating that the effect is a κ-receptor mediated event. The effect was abolished by pretreatment with ryanodine, a drug that mobilizes calcium from the sarcoplasmic reticulum. It was, however, not affected by nifedipine, a calcium antagonist or removal of external calcium. The results indicate that the increase in [Ca] i due to κ-opioid receptor stimulation results primarily from mobilization of calcium from an intracellular pool.

Measurements of intracellular calcium and contractility in human ciliary muscle

Electromechanical and pharmacomechanical coupling was investigated in human ciliary muscle by measuring the intracellular free calcium in single cultured ciliary muscle cells and the contractility in meridional ciliary muscle strips. The basal resting calcium concentration was 75 +/- 8.7 nmol/l, n = 23. Application of acetylcholine (0.1 mmol/l) and carbachol (0.1 mmol/l) resulted in an initial [Ca2+]i peak followed by a recovery phase and a [Ca2+]i plateau. The initial [Ca2+]i peak was still observed in the absence of extracellular calcium and in the presence of verapamil (0.1 mmol/l). During its plateau [Ca2+]i was decreased by withdrawal of extracellular calcium or application of verapamil (0.1 mmol/l). Depolarization induced by a high level of extracellular potassium yielded only a small transient [Ca2+]i peak without a [Ca2+]i plateau. In isolated ciliary muscle strips, muscarinic stimulation (carbachol 0.1 mmol/l) resulted in an initial phasic and a subsequent tonic contraction. Removal of external calcium reduced the phasic contraction to 30.6 +/- 4.4% (n = 8) and completely abolished the tonic one. Verapamil (0.1 mmol/l) had only a slight relaxing effect when applied during the tonic contraction. We conclude that human ciliary muscle contraction is mediated by calcium release from intracellular stores and calcium entry through calcium channels, which are most probably receptor-operated. Depolarization of the muscle cell membrane and calcium entry through voltage-operated calcium channels do not contribute significantly to human ciliary muscle contraction.

Voltage-gated currents in identified rat olfactory receptor neurons

Whole-cell recording techniques were used to characterize voltage-gated membrane currents in neonatal rat olfactory receptor neurons (ORNs) in cell culture. Mature ORNs were identified in culture by their characteristic bipolar morphology, by retrograde labeling techniques, and by olfactory marker protein (OMP) immunoreactivity. ORNs did not have spontaneous activity, but fired action potentials to depolarizing current pulses. Action potentials were blocked by tetrodotoxin (TTX), which contrasts with the TTX-resistant action potentials in salamander olfactory receptor cells (e.g., Firestein and Werblin, 1987). Prolonged, suprathreshold current pulses evoked only a single action potential; however, repetitive firing up to 35 Hz could be elicited by a series of brief depolarizing pulses. Under voltage clamp, the TTX-sensitive sodium current had activation and inactivation properties similar to other excitable cells. In TTX and 20 mM barium, sustained inward current were evoked by voltage steps positive to -30 mV. This current was blocked by Cd (100 microM) and by nifedipine (IC50 = 368 nM) consistent with L-type calcium channels in other neurons. No T-type calcium current was observed. Voltage steps positive to -20 mV also evoked an outward current that did not inactivate during 100-msec depolarizations. Tail current analysis of this current was consistent with a selective potassium conductance. The outward current was blocked by external tetraethylammonium but was unaffected by Cd or 4-aminopyridine (4-AP) or by removal of external calcium. A transient outward current was not observed. The 3 voltage-dependent conductances in cultured rat ORNs appear to be sufficient for 2 essential functions: action potential generation and transmitter release. As a single odorant-activated channel can trigger an action potential (e.g., Lynch and Barry, 1989), the repetitive firing seen with brief depolarizing pulses suggests that ORNs do not integrate sensory input, but rather act as high-fidelity relays such that each opening of an odorant-activated channel reaches the olfactory bulb glomeruli as an action potential.

Effect of thrombin on calcium homeostasis in chick embryonic heart cells. Receptor-operated calcium entry with inositol trisphosphate and a pertussis toxin-sensitive G protein as second messengers.

Thrombin increases intracellular calcium ([Ca++]i) in several cell types and causes a positive inotropic effect in the heart. We examined the mechanism of the thrombin-induced [Ca++]i increase in chick embryonic heart cells loaded with the fluorescent calcium indicator, indo-1. Thrombin (1 U/ml) increased both systolic and diastolic [Ca++]i from 617 +/- 62 and 324 +/- 46 to 1041 +/- 93 and 587 +/- 38 nM, respectively. An initial rapid [Ca++]i increase was followed by a more sustained increase. There were associated increases in contraction strength, beat frequency, and action potential duration. The [Ca++]i increase was not blocked by tetrodotoxin or verapamil, but was blocked by pretreatment with pertussis toxin (100 ng/ml). The thrombin-induced [Ca++]i increase was partly due to intracellular calcium release, since it persisted after removal of external calcium. The [Ca++]i increase in zero calcium was more transitory than in normal calcium and was potentiated by 10 mM Li+. Thrombin also induced influx of calcium across the surface membrane, which could be monitored using Mn++ ions, which quench indo-1 fluorescence when they enter the cell. Thrombin-induced Mn++ entry was insensitive to verapamil, but was blocked by 2 mM Ni++. Thrombin increased inositol trisphosphates by 180% at 90 s and this effect was also blocked by pretreatment with pertussis toxin. thrombin promotes calcium entry and release in embryonic heart cells even when action potentials are inhibited. Both modes of [Ca++]i increase may be coupled to the receptor by pertussis toxin-sensitive G proteins.

The C-fibre conduction block caused by capsaicin on rat vagus nerve in vitro

Capsaicin (0.01–50 μM) was applied to adult rat vagus nerve in vitro to examine the C-fibre conduction block and to compare its time course with that of the depolarisation caused by this drug. The conduction block was assessed by the reduction in the C-wave of the compound action potential. Capsaicin caused a dose-dependent decrease in the height and area under the C-wave; the threshold dose was between 0.01 and 0.3 μM and maximum C-wave reduction of about 85% occurred with doses of 5 μM or above. The C-wave reduction was divided into reversible and irreversible components which differed in dose dependency. The threshold for the reversible block was below 0.3 μM and for the irreversible block it was about 1 μM. The onset of the block took about 5 min, regardless of dose. Where there was more than 50% recovery after the block the reversible component was measured. This was dose dependent and its duration was 10–90 min. Removal of external calcium did not affect the magnitude of the C-wave block, although it did seem to prevent recovery of the C-wave.

Increased sodium-calcium exchange in arterial smooth muscle of spontaneously hypertensive rats.

We compared sodium-calcium (Na-Ca) exchange in vascular smooth muscle between spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Aortic rings of 11 SHR and 11 WKY rats aged 11-12 weeks were superfused with physiological saline, and isometric tension was measured. Systolic blood pressure was higher in SHR (174 +/- 12 mm Hg) than in WKY rats (132 +/- 4 mm Hg): 1) In the presence of 10 microM phentolamine, 10 microM verapamil, and 5 mM caffeine, reduction of ionized extracellular sodium concentration [( Na+]o) from normal (139.2 mM) to 1.2 mM (replaced by N-methyl-D-glucamine) caused an external Ca2+-dependent increase in tonic tension (calcium entry by Na-Ca exchange). The rate of increase was higher in SHR (35.4 +/- 3.9 mg/min) than in WKY rats (17.9 +/- 1.3 mg/min) (p less than 0.01). 2) In the presence of phentolamine, verapamil, and caffeine, relaxation from low-Na+ contraction was promoted by external calcium removal. The rate of relaxation was directly related to [Na+]o. The rates of relaxation at normal (139.2 mM) [Na+]o were higher in SHR than in WKY rats (p less than 0.05). The rates of relaxation at 1.2 mM [Na+]o (calcium extrusion by adenosine triphosphate-driven calcium pump) were not different between SHR (11.6 +/- 2.8 mg/min) and WKY rats (8.9 +/- 2.5 mg/min). The increase in the rates of relaxation from 1.2 mM to normal (139.2 mM) [Na+]o (calcium extrusion by Na-Ca exchange) was greater in SHR (34.9 +/- 6.6 mg/min) than in WKY rats (17.1 +/- 4.5 mg/min) (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Complement Proteins C5b-9 Induce Secretion of High Molecular Weight Multimers of Endothelial von Willebrand Factor and Translocation of Granule Membrane Protein GMP-140 to the Cell Surface

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture.

Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of -70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to -70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 microM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.