Free Tyrode Research Articles

Influence of the Formamidine Pesticide Amitraz and Its Metabolites on Porcine Myometrial Contractility - Involvement of α 2-Adrenoceptors and Ca 2+ Channels

Isolated uterine strips were used to study the effects of amitraz and its metabolites on porcine myometrial contractility during the luteal phase of the estrous cycle. Amitraz and its active metabolite BTS27271 (10 −8-10 −5 M) caused a dose-dependent increase in myometrial contractility in the luteal phase strips, and BTS27271 was more efficacious than amitraz in this aspect. The other metabolites of amitraz at less than or equal to 10 −4 M did not affect porcine myometrial contractility. The α 2-adrenoceptor antagonist, yohimbine (10 −8-3 × 10 −7 M), but not the α 1-adrenoceptor antagonist, prazosin (10 −6 M), blocked the effect of amitraz and BTS27271 in a dose-dependent manner. When uterine strips were pretreated with the Ca 2+-free Tyrode′s solution or the voltage-dependent Ca 2+ channel blocker verapamil (3 × 10 −5 M), the contractile effects of amitraz and BTS27271 were completely abolished, whereas those of carbachol were reduced. The results suggested that the amitraz- and BTS27271-induced myometrial contractions are mediated by α 2-adrenoceptors and this effect is mediated by an increase in extracellular Ca 2+ influx through voltage-dependent Ca 2+ channels.

Depressive effects of arenobufagin on the delayed rectifier K + current of guinea-pig cardiac myocytes

The effects of a bufadienolide isolated from toad venom, arenobufagin, a potent Na +/K + pump inhibitor, were studied in single guinea-pig ventricular cells in the whole-cell patch-clamp configuration. Arenobufagin (50 μM) applied extracellularly decreased the amplitude of the delayed rectifier K + current ( I dK) by 30% without affecting the gating kinetics. The L-type Ca 2+ current was also depressed, but to a lesser extent. The inward rectifier K + current was hardly affected. Ouabain and the internal dialysis of cells with the solution containing 20 mM Na + depressed I dK in a similar way as arenobufagin. On the other hand, arenobufagin also depressed I dK when the Na +/K + pump was already inhibited in the K +-free Tyrode solution. Therefore, both a direct effect on the channel and an indirect effect through the inhibition of the Na +/K + pump may be involved in the depression of I dK by arenobufagin.

Comparison of Phorbol Ester-induced Responses in Rabbit and Human Platelets and Difference in Shape Change

Phorbol 12,13-dibutyrate (PDBu) induced aggregation and ATP release of washed human and rabbit platelets in a concentration-dependent manner. The EC(50) for PDBu-induced aggregation of washed human and rabbit platelets were 67.6 ± 3.3 and 32.7 ± 5.9 nM, respectively. PDBu and l-oleoyl-2-acetylglycerol (OAG) also caused a shape change in rabbit platelets detected by microscopic and turbidimetric techniques. Both 20K and 47K proteins of rabbit platelets were phosphorylated by PDBu treatment. All the PDBu-induced responses of human and rabbit platelets were blocked by a protein kinase C inhibitor, staurosporine, in a concentration-dependent manner. In both human and rabbit platelets, PDBu induced no phosphoinositide breakdown, caused no increase of [Ca(2+)](f) and induced no formation of TxB(2). Indomethacin, BN52021, nitroprusside and cytochalasin B did not inhibit, whereas verapamil, prostaglandin E, and EGTA inhibited PDBu-induced aggregation of washed human and rabbit platelets. Apyrase and creatine phosphate/creatine phosphokinase inhibited PDBu-induced aggregation, but did not affect the shape change or 20K myosin light chain phosphorylation of rabbit platelets. In Ca(2+)-free Tyrode's solution containing 1 mM EGTA, PDBu induced a shape change in rabbit platelets and this effect was inhibited by staurosporine and cytochalasin B. It is concluded that PDBu and OAG induce a shape change in rabbit platelets probably through phosphorylation of 20K myosin light chain.

Effect of catecholamines on intracellular pH in sheep cardiac Purkinje fibres.

1. It has been reported that catecholamines affect intracellular pH (pHi) in a number of tissues, generally by altering the kinetics of the Na(+)-H+ exchanger. We postulated that catecholamines might affect pHi in cardiac tissue. We tested this in resting sheep cardiac Purkinje fibres by measuring transmembrane potential and pHi with standard and H(+)-sensitive microelectrodes. 2. Adrenaline and the beta-adrenergic agonist isoprenaline, both 5.0 x 10(-6) M, resulted in depolarization and intracellular acidification (adrenaline, 0.03 +/- 0.01 pH units, n = 8, P = 0.005; isoprenaline, 0.08 +/- 0.01 pH units, n = 17, P = 0.0001). The alpha-adrenergic agonist phenylephrine, at concentrations up to 200 microM, had no significant effect on membrane potential or pHi. 3. Isoprenaline significantly attenuated the half-time (t0.5) for pHi recovery from intracellular acidification induced via the NH4Cl pulse technique. Isoprenaline also attenuated the hyperpolarization that is normally seen at the onset of pHi recovery. Phenylephrine slightly reduced the t0.5 for recovery, although the reduction did not reach statistical significance. 4. Forskolin, 7.5-10 x 10(-5) M, an agent that raises intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP), also induced depolarization and acidification, similar to that induced by adrenaline and isoprenaline. 5. In the presence of the Na(+)-H+ exchange blocker 5-dimethyl amiloride, 2-6 x 10(-5) M, isoprenaline-induced acidification was blunted but not abolished. When administered in Na(+)-free Tyrode solution, isoprenaline-induced acidification was also not abolished. Buffering power, tested using the NH4Cl method, was not decreased by isoprenaline, but rather, was slightly increased. Reversal of H+ driving force across the cell membrane from the normally inward direction to outward (achieved by increasing pHo to 8.3-8.5 and depolarizing the membrane with 10 mM K+ solutions) did not prevent intracellular acidification from occurring in the presence of isoprenaline. When glycolysis was inhibited by a 60 min exposure to glucose-free solution containing 5.5 mM 2-deoxyglucose, acidification by isoprenaline was nearly abolished. 6. We conclude that, in resting sheep Purkinje fibres, beta- but not alpha-adrenergic stimulation results in intracellular acidification and depolarization, probably mediated via an increase in cyclic AMP. beta- but not alpha-adrenergic stimulation slows the rate of recovery from intracellular acidification and blunts the hyperpolarization associated with this recovery. 7. The intracellular acidification appears to be due both to partial inhibition of Na(+)-H+ exchange and to stimulation of glycolysis by beta-adrenergic agents.

Interdependence of intracellular taurine and sodium in guinea pig heart.

The aim was to investigate the effects of raising intracellular taurine on the intracellular sodium activity (aNa1) in isolated guinea pig ventricular myocytes, and the effect of procedures that raise intracellular sodium on taurine concentration in the perfused guinea pig ventricular tissue. Taurine was introduced into the sarcoplasm of isolated ventricular myocytes, either during cell isolation or by diffusion from a penetrating micropipette, and the effect on aNai was measured using an ion sensitive microelectrode. Guinea pig hearts, mounted on a Langendorff apparatus, were perfused with a variety of physiological media and the level of taurine in the ventricles determined using high pressure liquid chromatography. An increase in intracellular taurine caused by its presence during cell isolation or by diffusion from a micropipette significantly reduced the aNai of isolated myocytes at rest, during perfusion with Ca depleted solutions, or on inhibition of the Na pump. In the guinea pig ventricles, taurine at 13.0(SEM 0.6) mmol.kg-1 wet weight comprised up to 45% of the free amino acids; since plasma taurine was 64(13) mumol.litre-1, this means that in vivo a large outwardly directed gradient for taurine exists (equivalent to a free energy of 13.7 KJ.mol-1). Upon perfusion with Ca,Mg free Tyrode solution (which raises intracellular sodium markedly), a time dependent loss of taurine occurred. Both the rate of loss and the total amount lost were increased when the Na pump was also inhibited. This loss of tissue taurine was not due to release from dead or lysed cells, as it was antagonised by procedures known to reduce the rise of aNai during Ca depletion, was inhibited by beta alanine (an inhibitor of taurine transport), and the fall in tissue taurine was not correlated with the appearance of lactate dehydrogenase in the effluent. The data from isolated myocytes and perfused guinea pig hearts were consistent with the presence of a Na/taurine symport which is activated to cause efflux of Na and taurine when either rise above their physiological level.

Removal of extracellular Mg2+ suppresses sulfation of glycoconjugates secreted from rabbit trachea in culture.

The influences of extracellular Ca2+ and Mg2+ concentrations on the basal secretion of glycoconjugates from rabbit trachea in organ culture were examined. Over 80% of the 35S-labeled and [3H]glucosamine-labeled glycoconjugates secreted by the trachea were digested upon incubation with chondroitinase ABC. The basal secretion did not occur in the medium at 4 degrees C, indicating an energy-dependent process. The basal secretion at 37 degrees C of 35S-labeled glycoconjugates was prominently suppressed in Mg(2+)-free Tyrode solution but not in Ca(2+)-free Tyrode solution containing ethyleneglycol bis(2-aminoethylether)tetraacetic acid (EGTA). In contrast, the basal secretion of [3H]glucosamine-labeled glycoconjugates was not affected by the Mg2+ concentration in the medium. The results suggest that extracellular Mg2+ largely contributes to sulfation of glycoconjugates basally secreted from rabbit trachea.

Removal of Extracellular Mg2+ Suppresses Sulfation of Glycoconjugates Secreted from Rabbit Trachea in Culture

The influences of extracellular Ca2+ and Mg2+ concentrations on the basal secretion of glycoconjugates from rabbit trachea in organ culture were examined. Over 80% of the 35S-labeled and [3H]glucosamine-labeled glycoconjugates secreted by the trachea were digested upon incubation with chondroitinase ABC. The basal secretion did not occur in the medium at 4°C, indicating an energy-dependent process. The basal secretion at 37°C of 35S-labeled glycoconjugates was prominently suppressed in Mg2+-free Tyrode solution but not in Ca2+-free Tyrode solution containing ethyleneglycol bis(2-aminoethylether)tetraacetic acid (EGTA). In contrast, the basal secretion of [3H]glucosamine-labeled glycoconjugates was not affected by the Mg2+ concentration in the medium. The results suggest that extracellular Mg2+ largely contributes to sulfation of glycoconjugates basally secreted from rabbit trachea.

Inhibitory effect of lead on 5-hydroxytryptamine induced contractions of isolated ileum of rat

The present study examined the effects of lead (Pb) on the 5-hydroxytryptamine (5-HT) contracture and calcium (Ca++) kinetics of ileum of rat. Isolated ileum preparations incubated in excess Ca++ (2.8 mM) or in Ca++ free Tyrode solution showed decrease in sensitivity to 5-HT. The inhibitory action of Pb reduced the sensitivity and decrease maximum responses to 5-HT in any Mg++ concentration of the medium. In the normal Ca++ and its deficient medium deterioration in sensitivity to Pb and the relevant maximum response to 5-HT were observed. In excess Ca++, the Pb effect depressed corresponding to its maximal response to 5-HT. The presence of EDTA depressed the Pb effect irrespective of the Ca++ influence on the receptors. Thus Pb may be inhibiting responses to 5-HT by interfering Ca++ utilization.

Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Tranilast on Endothelin-Induced Bronchoconstriction in Guinea Pigs.

Tranilast, an anti-asthmatic drug with anti-allergic properties, inhibited endothelin (ET)-induced asthmatic like respiratory obstruction in guinea pigs when administered orally at 200 mg/kg 1 h prior to the intravenous injection of ET. ET also caused a bronchoconstriction detected as an increase in inflation pressure in Konzett-Rössler apparatus. Tranilast (200 mg/kg, p.o. 1 h before ET) inhibited ET-induced increased inflation pressure. ET-induced bronchoconstriction detected as an increase in inflation pressure was clearly inhibited by the administration of indomethacin (used as a reference drug) at doses of 1 and 5 mg/kg 30 min prior to onset of the reaction. In addition to the above in vivo experiments, tranilast at concentrations between 10(-5) and 10(-4) g/ml inhibited ET-induced contraction of isolated guinea pig tracheal muscle, whereas indomethacin did not affect this in vitro response. In order to elucidate the inhibitory mechanism of tranilast on ET-induced bronchoconstriction, the effects in Ca(2+)-free medium and on ET-induced histamine and prostaglandin F2 alpha release from tracheal muscle were investigated. Consequently, tranilast did not affect ET-induced contraction of guinea pig tracheal muscle in Ca(2+)-free Tyrode's solution and ET induced neither histamine nor PGF2 alpha release. These results suggest that tranilast inhibits ET-induced bronchoconstriction by inhibiting the ET-induced calcium influx into tracheal muscle.

The effects of phorbol esters on choline phospholipid hydrolysis in heart and brain

The efflux of choline was determined in rat striatal slices, incubated chicken atria and perfused chicken hearts. 4β-Phorbol-12β.13α-dibutyrate (PDB) and 4β-phorbol-12β-myristate.13α-acetate (PMA) were used to stimulate protein kinase C. The other phorbol esters, 4β-phorbol-13α-acetate (PAc) and 4α-phorbol-12β.13α-didecanoate (4αPDD), known to be inactive, were tested to evaluate the specificity of the responses. PDB markedly enhanced the efflux of choline in all of the three preparations. The PDB-evoked efflux of choline in incubated chicken atria was equal to the net production of choline and, therefore, was not caused by translocation of intracellular free choline. After inhibition of the cholinesterase activity, PDB linearly increased the efflux of choline in rat striatal slices, but failed to alter the spontaneous efflux of acetylcholine. Thus acetylcholine did not serve as the source of the PDB-evoked efflux of choline. PMA was as effective as PDB, whereas PAc and 4αPDD failed to alter the choline efflux in the perfused heart. Both infusion of a Ca 2+-free EGTA-containing Tyrode solution and mepacrine reduced the spontaneous efflux of choline by about 40% and blocked the PDB-evoked efflux of choline. In contrast, a Ca 2+-free solution without EGTA failed to alter the spontaneous and the PDB-evoked choline efflux. It is concluded that phorbol esters stimulate the hydrolysis of choline-containing phospholipids in heart and brain via activation of protein kinase C.

Evaluation of Calcium-Free Tyrode's Sperm Capacitation Medium for Use in Bovine In Vitro Fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10µg/ml),and estradiol-17β (1.5µg/ml) for 24h at 37°C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4h in Ca++-free Tyrode's medium (pH 7.6), were diluted to 2×106 sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 µ1 droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8h before transfer to fresh medium and then incubated for 40h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca++-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca++-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.

Effect of Washing and Capacitating Media pH on Bull Sperm Motility, Acrosome Integrity, and Ability to Penetrate Zona-Free Hamster Oocytes

Bovine-ejaculated sperm were washed thrice in bovine serum albumin-saline media, pH 7.2 to 8.4, and incubated at 37°C in Ca++-free Tyrode's media, pH 7.2 to 8.4, for 0, 2, 4, 6, and 8h. Motility was highest when sperm were washed in pH 7.2 medium and incubated in pH 8.0 or 8.4 media. Motility remained above 50% until 8h. Washing in pH 7.6, 8.0, or 8.4 media induced more acrosome reactions after incubation than washing at pH 7.2. Percentage of acrosome-reacted sperm increased at each successive time period. Sperm penetrated more oocytes at 4, 6, and 8h when wash medium pH was fixed at 7.2 and capacitating media pH was raised at .4 unit increments from 7.2 to 8.4. When sperm were washed in pH 7.2 medium, the postincubation penetration rates peaked at 8h. With wash media of pH 7.6, 8.0, or 8.4, the postincubation penetration rates peaked at 4h and then gradually declined. In conclusion, the most effective system for capacitating bull sperm was a pH 7.6 wash followed by capacitation in pH 7.6 medium for 4 to 8h and this system resulted in the highest penetration rates. Wash media pH hastened capacitation but was not a capacitating agent.

Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos.

As an outgrowth of in-vitro fertilization and embryo transfer, detection of genetic and metabolic defects prior to implantation might be possible in the future. The objective for preimplantation diagnosis would be to sample a minimum of cell material of the conceptus for diagnosis prior to transfer. Different protocols for isolating individual blastomeres out of 2-cell mouse embryos were evaluated. 2-cell mouse embryos (from F1 hybrids C57B1 females x CBA males) were collected and the zona pellucida was removed by enzyme treatment (pronase), by exposure to Ca2+-Mg2+-free acid Tyrode (pH = 2.5) or by mechanical force. Individual blastomeres were obtained by exposure to an enzyme (pronase), to a chelating agent (EDTA-glycine mixture), to Ca2+-Mg2+-free PBS or after isolation by mechanical force. The biopsied blastomeres were then cultured in vitro as such or first replaced into a host zona pellucida. Evaluation was performed by culture in vitro up to the blastocyst stage and by transfer of embryos appearing morphologically normal into pseudopregnant foster mothers. A chromosomal study of the second mitotic division of the isolated blastomere was also performed. All isolation procedures had a negative impact on the in-vitro and in-vivo growth patterns of the isolated blastomeres. After culture in vitro to the blastocyst stage, different abnormalities could be observed: embryos lacking compaction, embryos with double blastocoelic cavities and embryos with no inner cell mass (trophoblastic vesicle). After replacement of the isolated blastomeres into a host zona pellucida, similar observations could be made. Chromosomal analysis did not reveal a clear influence of the different biopsy methods on the mitosis of the isolated blastomeres.

Calcium‐activated non‐selective cation channel in ventricular cells isolated from adult guinea‐pig hearts.

1. A class of Ca2+-activated non-selective cation channel was identified in ventricular cells, which were dissociated from adult guinea-pig hearts using collagenase. 2. Under cell-attached conditions the patch electrode filled with a Na+-rich solution recorded no obvious single-channel current at the resting membrane potential. Subsequent superfusion of the ventricular cell with a Na+-free Tyrode solution induced an inward-going single-channel current as well as contracture of the cell. Kinetics of this channel were not affected by varying the membrane potential. 3. Single-channel currents showing a conductance similar to those observed in the cell-attached patches were recorded in isolated inside-out membrane patches when the inner side of the membrane was exposed to a free Ca2+ concentration ([Ca2+]i) higher than 0.3 microM. The slope conductance of the channel was 14.8 +/- 2.9 pS (mean +/- S.D., n = 17) at 20-25 degrees C. 4. The reversal potential examined in the inside-out patch was about 0 mV irrespective of the Na+-rich, K+-rich, Li+-rich or Cs+-rich solutions on either side of the membrane, thereby indicating that the channel was almost equally permeable to these cations. 5. The open probability of the channel was increased by raising [Ca2+]i with the maximum value of 0.93 +/- 0.17 (n = 4) at about 10 microM [Ca2+]i. The dose-response relation was fitted to the saturation kinetics with a Hill coefficient of 3.0 and a half-maximum concentration of 1.2 microM [Ca2+]i. 6. The gating kinetics were complex; both the open and closed time histograms showed at least two exponential components with time constants of 3.8 +/- 1.3 ms and 140 +/- 110 ms for open time and 1.8 +/- 1.1 ms and 14.9 +/- 5.3 ms for closed time (n = 4) at 10 microM [Ca2+]i. Reduction of [Ca2+]i resulted in both a decrease of the time constant of the slow component in the open time histogram and an increase of the two time constants of the closed time histogram. 7. Contribution of the channel to the whole-cell current was discussed based on an estimation of the channel density, presumably about 0.04 approximately 0.4/microns 2. Maximum activation of the channel would produce 7.2 approximately 72 nS of membrane conductance, which would explain the reported magnitude of the Ca2+-mediated background conductance of the single myocyte. The channel may also contribute, at least in part, to the transient inward current which develops in Ca2+-overloaded cardiac cells.

Modulation of vascular dynamics by spontaneous contraction of smooth muscle in the isolated carotid artery of the rat.

Possible roles of smooth muscle contractions in the vascular dynamics were studied in the isolated rat large artery which has previously been shown to generate spontaneous rhythmic contractions depending upon the extracellular Ca2+. A cylindrical segment of the common carotid artery was superfused externally and perfused intraluminally by Tyrode solution, and the steady state intraluminal pressure (P)-volume (V) relationship of the vessel was obtained. The pressure buffering characteristic (Windkessel effect) of the vascular wall was evaluated by measuring the intraluminal pressure swing during application of external alternating pressure (semi-triangular pressure pulse, 0-120 mmHg, 300 cycle/min, "dynamic pressure load") to the intraluminal space. Effects on these vascular dynamics of inhibition of spontaneous smooth muscle contraction during superfusion of the preparation with nominally Ca2+-free Tyrode solution were investigated. Spontaneous rhythmic fluctuations of the intraluminal pressure (0.2-1.0 mmHg, 2-25 cycle/min), presumably due to contractions of vascular smooth muscles, were detected. The Ca2+-free solution abolished these spontaneous changes in the intraluminal pressure and reduced the baseline intraluminal pressure. The volume distensibility (delta V/(V X delta P] of the preparation at higher intraluminal fluid volumes (or pressures, higher wall stretch) was increased by Ca2+-free solution. The magnitude of intraluminal pressure swing and the maximum rate of changes in the pressure during application of "dynamic pressure load" were decreased in Ca2+-free solution. The results suggest that the inhibition of spontaneous contractions of smooth muscle by Ca2+-free solution may increase the volume distensibility and pressure-buffering function of the large elastic artery.

Ca 2+ transport systems mediating the high K +/Low Na +-induced uptake of Ca 2+ into rat atrium

The effect of high K+/low Na+-Tyrode's solution on Ca2+ uptake into neonatal rat atrium was studied using 45Ca2+. Substitution of 60-129 mM Na+ in Tyrode's solution by equimolar concentrations of K+ or choline, significantly (with the exception of 60 mM choline substitution) increased Ca2+ uptake above control. Furthermore, the Ca2+ uptake stimulated by K+ substitution was significantly greater than that stimulated by choline substitution at the corresponding concentrations. The choline/low Na+-induced Ca2+ uptake (i.e. that above the Ca2+ uptake measured in normal Tyrode's solution) was increased by pre-exposure to either ice-cold Tyrode's solution for 1 h (approximately 36% increase) or to K+-free Tyrode's solution for 3 h (approximately 100% increase). The choline/low Na+-induced Ca2+ uptake was abolished by the hypertonic addition of NaCl (returning the bathing Na+ concentration to normal), increased (approximately 140%) by the addition of 1.8 mM PO4(3-)-free Hepes buffered choline/low Na+ media, but unaffected by 0.2 mM cadmium. The high K+/low Na+-induced Ca2+ uptake (i.e. that above the Ca2+ uptake measured in normal Tyrode's solution) was relatively insensitive to pre-exposure to cold (0% change) or K+-free media (11% increase) and only 50% inhibited by the hypertonic addition of NaCl (returning the bathing Na+ concentration to normal). However, the high K+/low Na+-induced Ca2+ uptake was 57% inhibited by 0.2 mM cadmium and approximately 30% inhibited by the addition of 1.8 mM PO4(3-) to HCO3-/PO4(3-)-free Hepes buffered high K+/low Na+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle relaxant properties of the aqueous extract of portulaca oleracea

The skeletal muscle relaxant properties of an aqueous extract of Portulaca oleracea were examined on the twitch and tetanus tension evoked by electrical stimulation using the rat phrenic nerve-hemidiaphragm and frog sciatic nerve-sartorius muscle preparations and on contractures induced by nicotinic agonists using the rat rectus abdominis muscle preparation. The extract (5—50 × 10 −4 g ml ) produced a dose-dependent initial enhancement, followed by a longer lasting depression of twitch tension as induced by indirect electrical stimulation (NS) as well as direct stimulation (MS) of the diaphragm and sartorius muscle preparations. The augmentation of twitch amplitude (MS) produced by the extract was not significantly antagonised or potentiated by d-tubocurarine (5—50 × 10 −8 M) or physostigmine (5—50 × 10 −8 M) at concentrations which blocked or potentiated NS-induced twitch contractions. Furthermore, tetrodotoxin (5 × 10 −7 g ml ) alone or in combination with d-tubocurarine did not significantly attenuate extract-induced augmentation of twitch contractions to MS. In addition, the depression of twitch tension due to NS was not antagonised by physostigmine but was reversed and/or abolished by Ca 2+ (2—5 × 10 −3 M or potassium thiocyanate (1× 10 −3 M). Contractures induced by K + (80 mM) or by tetanic stimulation (20—60 Hz) were significantly reduced by the extract whereas, contracture induced by caffeine (2.5—6.0 mM) was not affected. Ca 2+-free Tyrode's solution and EDTA (1.25 × 10 −3 g ml ) potentiated extract-induced depression of twitch tension to MS. On the rectus abdominis, contractures induced by nicotinic agonists (acetylcholine, 3—500 × 10 −7 M; carbachol, 5—500 × 10 −7 M and nicotine, 5—500 × 10 −8 M were significantly attenuated and/or abolished by the extract (5—10 × 10 −3 g ml ). These observations indicate that the aqueous extract possesses unique skeletal muscle relaxant properties which do not appear to involve interference with cholinoceptor mechanism(s). It appears that the mechanism of action of the extract may involve interference with Ca 2+ mobilization in skeletal muscle.

Cellular electrophysiologic changes and “arrhythmias” during experimental ischemia and reperfusion in isolated cat ventricular myocardium

The cellular electrophysiologic consequences of both regional and global experimental ischemia and reperfusion were studied in the isolated cat myocardium, using conventional microelectrode techniques. Oxygenated Tyrode's solution was perfused through the left anterior descending and circumflex coronary arteries, while the preparation was superfused with Tyrode's solution gassed with 95% nitrogen and 5% carbon dioxide. Electrophysiologic characteristics of endocardial muscle cells were normal during coronary perfusion. When perfusion was discontinued for 30 minutes, resting membrane potential was decreased by 21.6 +/- 4.1%, action potential amplitude was decreased by 29.1 +/- 8.6% and action potential duration was decreased by 54.1 +/- 12.5% (p less than 0.001). Ectopic activity occurred after 5 to 10 minutes of ischemia and was more frequent in regional than in global ischemia (p less than 0.05). Rapid ventricular activity was observed in only 5 (17%) of 29 preparations during ischemia, whereas it occurred in 24 (83%) of 29 preparations during reperfusion. Rapid ventricular activity began 5 to 40 seconds (mean 19) after the start of reperfusion, stopped spontaneously after a mean of 113 +/- 211 seconds and occurred after both regional and global ischemia. The cellular electrophysiologic changes induced by ischemia returned to baseline values within the next 5 minutes. Repeated ischemia and reperfusion runs reproduced the same electrophysiologic changes and rapid ventricular activity. Coronary perfusion with procainamide (20 mg/liter) aggravated the ischemic depressions of action potential amplitude and action potential duration and increased conduction delay during ischemia, but it did not prevent rapid ventricular activity induced by reperfusion. In contrast, verapamil (1 mg/liter) perfusion did not affect the changes in action potential variables during ischemia but prevented reperfusion-induced rapid ventricular activity. Perfusion with calcium ion (Ca2+)-free Tyrode's solution just before ischemia and during reperfusion slowed or prevented reperfusion-induced rapid ventricular activity, without affecting the action potential changes during ischemia. It is concluded that, in these isolated perfused ventricular muscle preparations, different mechanisms may be operative in ischemic and reperfusion arrhythmias and Ca2+ may play an important role in the development of arrhythmias during the reperfusion phase of ischemia/reperfusion sequences.

Characterization of choline efflux from the perfused heart at rest and after muscarine receptor activation.

The resting efflux of choline from perfused chicken hearts varied from 0.4 to 2.6 nmol/g min, but was constant for at least 80 min in the individual experiments. The rate of choline efflux was found to be equal to the rate of choline formation in the heart, which, from the following reasons, was essentially due to hydrolysis of choline phospholipids. Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). Resting release of acetylcholine was 0.016 nmol/g min and, after inhibition of cholinesterase, only about 0.1 nmol/g min. Resting efflux of choline was reduced by mepacrine, a phospholipase A2 inhibitor, by perfusion with a Ca2+-free Tyrode's solution containing EGTA and by the combination mepacrine plus Ca2+-free/EGTA solution. In all experiments the reduced choline efflux levelled off within 10 min at about 50%. Omission or elevation of Mg2+ from 1.05 to 10.5 mmol/l had no effect. Resting efflux was increased to 150% by oleic acid (as sodium salt; 2 X 10(-5) mol/l) which is known to activate phospholipase D. Likewise muscarinic agonists (carbachol and acetylcholine) caused facilitation of the efflux of endogenous choline that was blocked by 3 X 10(-7) mol/l atropine. This effect was not reduced, but even slightly enhanced, by mepacrine and by infusion of EGTA in a modified Tyrode's solution (Ca2+-free, 10.5 mmol/l Mg2+). It is concluded that the resting efflux of choline from the heart is essentially due to hydrolysis of choline phospholipids, that half of the efflux is insensitive to mepacrine and is Ca2+-independent (excluding an involvement of phospholipase A2).(ABSTRACT TRUNCATED AT 250 WORDS)