Normal Krebs Solution Research Articles

Isoprenaline enhances both voltage-dependent Ba2+ and K+ currents in guinea-pig vas deferens.

In the guinea-pig vas deferens, isoprenaline (ISO) can exert significantly enhanced effects on voltage-dependent Ca2+ channel current carried by Ba2+ (IBa) via beta-adrenoceptor activation. Voltage-dependent Ba2+ (IBa) and K+ currents (I K) were recorded in the preparation voltage-clamped in a Krebs solution replaced Ca2+ by Ba2+ (Ba2+ solution) of 36 degrees C. At a holding potential of -40 mV, ISO markedly increased IBa evoked by depolarizing pulses, whereas it also increased I K at a holding potential of -20 mV so as to inactivate Ca2+ channel. This ISO-induced enhancement of IBa seems to be contradictory to the suppression of contraction by beta-adrenoceptor activation in normal Krebs solution.

Short term plasticity caused by Ca 2+ induced Ca 2+ release in the frog motor nerve terminals

The actions of ephedrine were studied using intracellular recording techniques in guinea pig intercostal muscle fiber. After application of ephedrine (10−5 −10−4 M), miniature end-plate potential (MEPP) amplitude was decreased in a concentration-dependent manner. A greater depression in amplitude of end-plate potentials (EPP) was observed in preparations immobilized with d-tuboeurarine. In addition, ephedrine depressed the transient depolarization produced by iontophoretically applied acetylcholine (ACh).In normal Krebs solution and high Mg2+ solution, 10−4 M ephedrine increased MEPP frequency but did not alter the quantal content of EPPs in high Mg2+ blocked muscle. In these preparations, EPP amplitude did not change significantly. These results suggested that in the absence of a postsynaptic receptor blocking agent, a slight presynaptic action of ephedrine may offset the decreased responsiveness of the postsynaptic membrane. In addition, no significant alterations of resting membrane potential or the electrical characteristics of the membrane were recorded.Pretreatment of a mouse phrenic nerve-diaphragm preparation with ephedrine did not protect against the irreversible blockade by α-bungarotoxin. These results suggested that ephedrine may depress ACh potentials, MEPPs, and EPPs in dTc-blocked muscle by acting postsynaptically at a site on or associated with the ACh receptor other than the ACh recognition site.

Extracellular sodium concentration has diverse effects on the hypoxia-induced increase in intracellular Ca 2+ in rat hippocampal slices

The intracellular free Ca 2+ concentration ([Ca 2+] i) in the CA1 pyramidal cell layer was measured using fura-2-loaded hippocampal slices prepared from adult rats. Hypoxia (oxygen-glucose deprivation) elicited a gradual increase in [Ca 2+] i in normal Krebs solution. With a high extracellular sodium concentration ([Na +] o), the hypoxia-induced response was attenuated, its onset-latency was longer and the time constant of its decay phase was shorter than in controls. In contrast, hypoxia in low [Na +] o elicited a significantly enhanced response with a short onset-latency and delayed decay phase. This exaggerated response to hypoxia in low [Na +] o was reversed by pre-incubation of the slice in low [Na +] o prior to the hypoxic insult. Some possible mechanisms and the functional significance of the observed effects of [Na +] o on the hypoxia-induced increase in [Ca 2+] i are discussed, with particular emphasis on the putative participation of the glutamate transporter and the sodium-calcium exchanger in hypoxia-induced neuronal injury.

Study of the in vivo and in vitro cardiovascular effects of a hydralazine-like vasodilator agent (HPS-10) in normotensive rats.

1. In this work, the cardiovascular effects of HPS-10, a new vasodilator agent, were studied in rats. 2. In conscious normotensive rats, oral administration of HPS-10 (4-9 mg kg-1) produced a dose-related and long-lasting fall in systolic arterial blood pressure (ED30 of 5.32 mg kg-1), accompanied by an increase in heart rate (ED30 of 8.43 mg kg-1). This tachycardia was totally inhibited by pretreatment with (+/-)-propranolol (10 mg kg-1, p.o.). 3. In anaesthetized normotensive rats, HPS-10 (0.3-0.6 mg kg-1, i.v.) produced a gradual, dose-dependent and sustained decrease in systolic, diastolic and mean arterial pressure (MAP) (ED30 for MAP of 0.41 mg kg-1, i.v.), accompanied by a significant bradycardia at high doses (> 0.4 mg kg-1; ED20 of 0.61 mg kg-1, i.v.). HPS-10 (0.5 mg kg-1, i.v.) did not modify the positive chronotropic effects induced by intravenous administration of noradrenaline (NA; 5 micrograms kg-1), angiotensin II (AII; 0.2 microgram kg-1) and nicotine (200 micrograms kg-1) but markedly inhibited the hypertensive response produced by these agents. 4. In rat isolated rubbed aorta, HPS-10 (0.1-1 mM) non-competitively and with almost equal effectiveness antagonized the contractions induced by NA, AII (in normal Krebs solution) and Ca2+ (in depolarizing Ca(2+)-free high-K+ 50 mM solution). In the experiments in Ca(2+)-free medium, HPS-10 (1 mM) considerably inhibited the contractions induced by NA, AII and caffeine in rat aorta. 5. Furthermore, in the studies with radioactive Ca2+, HPS-10 (1 mM) did not modify the basal uptake of 45Ca2+ but strongly decreased the influx of 45Ca2+ induced by NA, AII and K+ in rat aortic rings. 6. In rat isolated atria, HPS-10 (1 mM) produced a positive inotropic/negative chronotropic effect. 7. HPS-10 (0.3 mM) significantly inhibited the sustained and transient Ba2+ inward current (IBa) recorded in whole-cell clamped rat aortic myocytes. 8. These results indicate that the non-selective vasorelaxant effects of HPS-10 in rat aortic rings can be attributed to transmembrane Ca(2+)-antagonist activity and an intracellular action on smooth muscle cells. The direct vasodilator action of HPS-10 observed in rat isolated aorta may be responsible for the HPS-10 hypotensive activity in anaesthetized normotensive rats.

Vascular reactivity to endothelium-derived relaxing factor in human umbilical artery at term pregnancy

It has been reported that human umbilical artery (HUA) at term pregnancy released endothelium-derived relaxing factor (EDRF), using a superfusion bioassay system. However, other reports showed that endothelium-dependent relaxation was not observed in isometric tension studies using HUA ring with intact endothelium. Thus, we intended to clarify whether vascular smooth muscle of HUA at term is sensitive to EDRF. HUA was obtained after normal vaginal delivery or cesarean section at term. Isometric tension studies were performed in normal Krebs solution, using HUA rings or strips, which were prepared in calcium-free Krebs solution. Sodium nitroprusside (SNP), a nitric oxide (NO) donor drug, relaxed HUA rings precontracted with 0.1 microM 5-hydroxytryptamine (5HT) in a dose-dependent manner (1 nM-10 microM). Histamine, substance P, carbachol, or the calcium ionophore A23187, which are considered to be EDRF-releasing agents, did not relax the HUA rings. By immunohistochemical study, it was confirmed that endothelial cells were present in the luminal surface of the HUA rings after the isometric tension recording. In a co-axial bioassay system involving HUA strips denuded of endothelium and rabbit aorta with intact endothelium, HUA strips precontracted with 0.1 microM 5HT were relaxed in response to 1 microM SNP but not 1 microM carbachol, which released EDRF from the endothelium of rabbit aorta. These findings suggest that HUA at term is sensitive to NO but not EDRF.

Vascular reactivity to endothelium-derived relaxing factor in human umbilical artery at term pregnancy

It has been reported that human umbilical artery (HUA) at term pregnancy released endothelium-derived relaxing factor (EDRF), using a superfusion bioassay system. However, other reports showed that endothelium-dependent relaxation was not observed in isometric tension studies using HUA ring with intact endothelium. Thus, we intended to clarify whether vascular smooth muscle of HUA at term is sensitive to EDRF. HUA was obtained after normal vaginal delivery or cesarean section at term. Isometric tension studies were performed in normal Krebs solution, using HUA rings or strips, which were prepared in calcium-free Krebs solution. Sodium nitroprusside (SNP), a nitric oxide (NO) donor drug, relaxed HUA rings precontracted with 0.1 microM 5-hydroxytryptamine (5HT) in a dose-dependent manner (1 nM-10 microM). Histamine, substance P, carbachol, or the calcium ionophore A23187, which are considered to be EDRF-releasing agents, did not relax the HUA rings. By immunohistochemical study, it was confirmed that endothelial cells were present in the luminal surface of the HUA rings after the isometric tension recording. In a co-axial bioassay system involving HUA strips denuded of endothelium and rabbit aorta with intact endothelium, HUA strips precontracted with 0.1 microM 5HT were relaxed in response to 1 microM SNP but not 1 microM carbachol, which released EDRF from the endothelium of rabbit aorta. These findings suggest that HUA at term is sensitive to NO but not EDRF.

Superoxide and nitroglycerin stimulate release of PGF2 alpha and TxA2 in isolated rat heart.

The aim of this study was to investigate the effects of nitroglycerin (NTG), a nitric oxide (NO) donor used as a vasodilating agent, on prostanoid [e.g., prostaglandin (PG)] release in the O2(-)-pretreated rat heart. Perfusion of O2-, generated by a xanthine oxidase-purine coupling, caused elevation (P < 0.05) of the coronary perfusion pressure (CPP) after 20 min (from 57.1 +/- 3.9 during the control period to 72.2 +/- 3.9 mmHg, P < 0.05). O2- caused increased release of PGF2 alpha from 3.6 +/- 0.7 to 20.6 +/- 4.4 pmol.min-1.g-1 and of thromboxane A2 (TxA2) from 2.4 +/- 0.4 to 9.6 +/- 1.6 pmol.min-1.g-1 (P < 0.001) with no significant changes in PGE2 and PGI2 release. During the 20-min washout of O2- from the heart with normal Krebs solution, release of PGF2 alpha and TxA2 decreased to 8.7 +/- 1.4 and 6.3 +/- 1.7 pmol.min-1.g-1, respectively, and the release of PGE2 and PGI2 markedly increased from 11.1 +/- 2.9 to 25.4 +/- 3.6 and 157.2 +/- 16.4 to 413.2 +/- 41.4 pmol.min-1.g-1, respectively (P < 0.05), without lowering the elevated CPP. Administration of 4 microM NTG during the washout period paradoxically augmented the elevated CPP to 133.3 +/- 0.6% and was associated with a doubling (P < 0.05) of PGF2 alpha and TxA2 release with no significant changes in PGE2 and PGI2 release. The NTG-induced CPP elevation was inhibited (P < 0.05) by indomethacin, a cyclooxygenase inhibitor, or ONO-3708, a TxA2 receptor blocker, whereas arachidonic acid, a substrate for PG synthesis, augmented the CPP elevation. These results indicate that NTG stimulates the synthesis of vasoconstrictive PG in the O2(-)-pretreated rat heart, inducing a paradoxical elevation in CPP.

Pharmacological Actions of Naringin on Alpha, Beta-adrenoceptors and Uptake of Noradrenaline in Rat Isolated Vas Deferens

In normal Krebs solution, naringin (1×10−7 Msc–2×10−6 M) enhanced noradrenaline-induced contractions in rat isolated vas deferens increasing this potentiation in a concentration dependent manner. This enhancing effect continued in the presence of yohimbine (10−6 M) or propranolol (10−6 M). Naringin did not modify significantly the dopamine dose-response curves. In a medium containing 10−8 M prazosin, naringin decreased noradrenaline dose-response curves underneath the control curve at all the doses tested. Naringin did not modify significantly the NA dose-response curve in a medium containing cocaine (10−5 M) or oestradiol (10−5 M). The potentiation produced by naringin on the NA-induced contractions in rat vas deferens may be due to alpha-1 receptor activation, together with an interference of naringin in catecholamine uptake process.

Dopamine D1-like receptor-mediated presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones.

1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3.81 +/- 0.09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM). 2. Bath-applied dopamine (DA) reduced evoked EPSC amplitude by up to 54.2 +/- 2.3% with an IC50 of 19.9 microM in normal Krebs solution (2.5 mM Ca2+, 1.2 mM Mg2+) without effect on postsynaptic holding current. 3. DA (30 microM) reduced the mean frequency of spontaneous miniature EPSCs recorded in 0.5 microM tetrodotoxin without affecting their mean amplitude, rise time or decay time constant. This effect was diminished by 100 microM Cd2+. 4. The effect of DA on evoked EPSCs was mimicked by the D1-like receptor agonist, SKF 81297 (IC50 25.6 microM), but not by the D2-like receptor agonist R(-)-TNPA (30 microM) or (-)-quinpirole (30 microM), and was antagonized by the D1-like receptor antagonist R(+)-SCH 23390 (estimated dissociation constant KB = 1.7 microM) but not by the D2-like receptor antagonist S(-)-eticlopride (10 microM). 5. Forskolin (10 microM) reduced evoked EPSCs to approximately 60% of the control amplitude, and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D1-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

Evidence for mediation by endothelium‐derived hyperpolarizing factor of relaxation to bradykinin in the bovine isolated coronary artery independently of voltage‐operated Ca2+ channels

1. The role of endothelium-derived hyperpolarizing factor and voltage-operated Ca2+ channels in mediating endothelium-dependent, NG-nitro-L-arginine (L-NOARG; 100 microM) -resistant relaxations to bradykinin (BK), was examined in isolated rings of endothelium-intact bovine left anterior descending coronary artery. 2. Rings of artery were contracted isometrically to approximately 40% or their respective maximum contraction to 125 mM KCl Krebs solution (KPSSmax) with the thromboxane A2-mimetic, U46619. Relaxations to BK and the endothelium-independent NO donor, S-nitroso-N-acetylpenicillamine (SNAP), were normalized as percentages of reversal of the initial contraction to U46619. All experiments were carried out in the presence of indomethacin (3 microM). 3. BK caused concentration-dependent relaxations [sensitivity (pEC50) 9.88 +/- 0.05; maximum relaxation (Rmax), 103.3 +/- 0.5%] in U46619-contracted rings of bovine coronary artery. L-NOARG (100 microM) caused a significant (P < 0.01) 3 fold reduction in the sensitivity to BK (pEC50, 9.27 +/- 0.11) without affecting the Rmax (101.8 +/- 2.3%). A similar, significant 3 fold reduction in sensitivity to BK with no change in Rmax was observed after treatment with oxyhaemoglobin (20 microM; pEC50, 9.18 +/- 0.13, P < 0.001) or a combination of oxyhaemoglobin (20 microM) and L-NOARG (100 microM; pEC50, 9.08 +/- 0.10, P < 0.001). Oxyhaemoglobin (20 microM) either alone or in combination with L-NOARG (100 microM) caused an approximate 600 fold decrease in the sensitivity to SNAP. 4. The L-type voltage-operated Ca2+ channel inhibitor, nifedipine (0.3 microM-3 microM), reduced the maximum contraction (Fmax) to isotonic 68 mM KCl Krebs solution (103.5 +/- 2.0% KPSSmax) by 85-90% (P < 0.001); yet, the highest concentration of nifedipine (3 microM) caused only a small but significant reduction in both the sensitivity and Fmax to U46619. By contrast, nifedipine (3 microM) had no effect on the relaxation response to BK. Furthermore, a combination of nifedipine (3 microM) and L-NOARG (100 microM) had no further inhibitory effects on relaxations to BK (pEC50, 8.79 +/- 0.10; Rmax, 101.7 +/- 2.4%) than did L-NOARG (100 microM) alone (pEC50, 9.05 +/- 0.12; Rmax, 99.62 +/- 1.19). Also, nifedipine (0.3 microM and 3 microM) had no effect on the maximum relaxation to the K+ channel opener, levcromakalim (0.3 microM). 5. In the presence of nifedipine (0.3 microM to control contractions induced by high KCl) and isotonic 68 mM KCl Krebs solution (to inhibit K+ channel activity), relaxations to BK (pEC50, 9.42 +/- 0.10; Rmax, 93.9 +/- 1.8%) were similar to those observed in normal Krebs solution (pEC50, 9.58 +/- 0.09; Rmax, 98.4 +/- 0.8%). However, in the presence of 68 mM KCl Krebs solution the inhibitory effect of L-NOARG (100 microM) on relaxations to BK (pEC50, 8.53 +/- 0.20; Rmax, 31.0 +/- 11.3%) was markedly greater than that in normal KCl Krebs solution (pEC50, 9.12 +/- 0.08; Rmax, 91.5 +/- 2.0%). Similar treatment with 68 mM KCl Krebs had no effect on relaxations to the NO donor, SNAP, yet abolished the response to the K+ channel opener, levcromakalim (0.3 microM). 6. In summary, this study has shown that (1) NO synthesis in response to BK in bovine coronary artery endothelial cells in situ is likely to be abolished by L-NOARG, (2) NO-independent relaxations to BK are markedly attenuated by 68 mM KCl-containing Krebs, which, in the absence of L-NOARG, had no effect, (3) nifedipine blocked contractions to a maximum-depolarizing stimulus (KCl) yet had no effect on NO-independent relaxations to BK, and (4) maximum relaxations to levcromakalim were abolished by 68 mM KCl Krebs but were not affected by nifedipine. Therefore, we hypothesize that if smooth muscle hyperpolarization is involved in non-NO-, endothelium-dependent relaxation in bovine coronary arteries contracted with U46619, then it can accomplish this via a mechanism which does not i

Electrophysiological effects of nitric oxide in mouse superior mesenteric ganglion

Effects of nitric oxide (NO) generated from sodium nitroprusside (SNP) on neurons of mouse superior mesenteric ganglion (SMG) were studied in vitro using intracellular recording techniques. SNP solutions caused a membrane hyperpolarization in the majority (64%) of the neurons tested or a hyperpolarization followed by a depolarization in 8% of the neurons tested. The SNP-induced hyperpolarization persisted in a low-Ca2+ high-Mg2+ solution, indicating a direct effect of NO on the postsynaptic membrane. The hyperpolarizing effect of SNP was reduced or abolished by oxyhemoglobin. Electrical stimulation of the colonic nerves evoked a late slow excitatory postsynaptic potential (late sEPSP) in a population of neurons in normal Krebs solution. The amplitude of the late sEPSP was significantly enhanced in the presence of NG-nitroL-arginine, a NO synthase inhibitor. The results, particularly those observed with the NO synthase inhibitor, suggest that endogenous NO was released in the mouse SMG by repetitive nerve stimulation and that it modulated slow synaptic transmission, presumably by a direct action on ganglionic neurons.

Mechanisms of ionic currents involved in suppressive effect of isoprenaline on the action potential of guinea-pig vas deferens in normal Krebs solution.

The effects of beta-adrenoceptor stimulation by isoprenaline (ISO) on action potential and membrane current were studied in the guinea-pig vas deferens in normal Krebs solution by using a double sucrose gap method. In current-clamp experiments, ISO produced the membrane hyperpolarization by reducing the resistance and modified the pattern of multi-spike activity elicited by long depolarizing currents; an initial spike potential was reduced in the amplitude and the rate of rising phases and the slope of diastolic depolarization was slowered, leading to a prolongation of the spike discharge interval. In voltage-clamp experiments, ISO greatly enhanced the late outward K+ current (Ik2) by increasing the conductance, but hardly affected the peak outward K+ current (Ik1). ISO also reduced the inward Ca2+ current (Ica) by decreasing the conductance (ga) as well as by reducing the driving force for Ca2+. While it increased the leakage conductance (g1) due to K+ associated with hyperpolarizing voltage steps. In the preparations treated with Ca2+ channel blocker such as D-600, the enhanced effect of ISO on the Ik was still prominent, suggesting that Ca2+ and K+ channels appear to be independently regulated during beta-adrenoceptor stimulation. These results suggest that ISO exerts depressant actions on the vas deferens muscle by reduction of the Ica as well as by prominent enhancement of the Ik2 via beta-adrenoceptor activation.

The role of diacylglycerol and activation of protein kinase C in alpha 1A-adrenoceptor-mediated contraction to noradrenaline of rat isolated epididymal vas deferens.

1. The mechanism of contraction to noradrenaline (pEC50 5.6 +/- 0.1) in the rat epididymal vas deferens (mediated via alpha 1A-adrenoceptors) has been studied in functional experiments. 2. Contractions to noradrenaline at 10(-6) M were potentiated by the diacylglycerol (DAG) kinase inhibitor R 59022 (3 x 10(-7) M) from 49 +/- 4% to 63 +/- 3% maximum response and the time taken from initiation of contraction to the maximum response was reduced from 16 +/- 2 s to 9 +/- 1 s. The same contractions were not significantly potentiated by the DAG lipase inhibitor, U-57,908, 10(-5) M (51 +/- 2% control and 53 +/- 4% in the presence of U-57,908) nor was the time taken from initiation of contraction to the maximum response significantly altered (17 +/- 1 s control and 16 +/- 1 s in the presence of U-57,908). 3. Concentration-dependent contractions to noradrenaline (NA) were reduced by staurosporine (10(-7) M) and the selective protein kinase C inhibitor, calphostin C (10(-6) M) from 68 +/- 2% (NA, 3 x 10(-6) M) to 28 +/- 2% and 20 +/- 2% respectively and from 94 +/- 2% (NA, 3 x 10(-5) M) to 50 +/- 2% and 44 +/- 2% respectively. Contractions to K+ (40 +/- 2% maximum response to NA) were also significantly reduced by staurosporine (10(-7) M) (35 +/- 2%) but not by calphostin C (43 +/- 3%). 4. The phorbol ester, phorbol-12,13-dibutyrate (PDBu), produced a phasic, concentration-dependent contraction (10(-7) M - 10(-4) M) which was 41 +/- 2% of the maximum response to NA at 10(-4) M PDBu. The contraction to PDBu (10(-5) M) was reduced by calphostin C (10(-6) M) from 33 +/- 5% to 4 +/- 1% maximum response to NA. 5. Non-cumulative contractions to NA (10(-8) M - 10(-4) M) were abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) and were reduced in the presence of nifedipine (10(-6)M) in normal Krebs solution by 91 +/- 2% at 10(-4)M NA. The contraction to PDBu (10(-5)M, 33 +/- 5% maximum response to NA) was also abolished in Ca(2+)-free Krebs solution containing EGTA (1 mM) or by the presence of nifedipine (10(-6)M) in normal Krebs solution. 6. When NA (10(-4)M) was added to vasa deferentia in Ca(2+)-free Krebs solution containing EGTA (1 mM), following its wash out (and with EGTA later removed from the Krebs solution), readdition of Ca2+ (2.5 mM) to the Krebs solution produced no response. Cyclopiazonic acid (10(-5)M), which can deplete Ca2+ from intracellular stores, also produced no contraction. Therefore influx of extracellular Ca2+ is not a consequence of depletion of intracellular Ca2+ stores (capacitative Ca2+ influx). 7. Pre-incubation of tissues for 30 min with either cyclopiazonic acid (10(-5)M) or ryanodine (10(-4)M), which can both deplete intracellular Ca2+ stores, did not reduce the contractions to NA (3 x 10(-6)M). Pre-incubation of vasa deferentia with cyclopiazonic acid (1 or 3 min, when any rise in [Ca2+]i produced by cyclopiazonic acid might still exist) did not potentiate the contraction to PDBu (10(-5)M). Thus mobilization of intracellular Ca2+ may not be required for the activation of protein kinase C involved in these contractions. 8. In conclusion, the contraction of the rat epididymal vas deferens to NA mediated by alpha 1A-adrenoceptors appears to depend upon activation of protein kinase C by diacylglycerol, resulting in the influx of extracellular Ca2+ through voltage-gated Ca2+ channels. There was no evidence for a role of inositol trisphosphate in the contraction to noradrenaline in this tissue.

Enhancement by dibutyryl cyclic AMP of voltage-dependent Ca2+ and K+ currents in the guinea-pig vas deferens.

This study was designed to investigate a possible role for intracellular cyclic AMP involved in agonist-induced changes in electrical activity of smooth muscle of the guinea-pig vas deferens. The action of dibutyryl adenosine 3', 5'-phosphate (dibutyryl cyclic AMP) (up to 30 microM) was examined in current- and voltage-clamp, using the double sucrose gap method. Under current-clamp, dibutyryl cyclic AMP clearly shortens the duration of action potential by hastening the rates of depolarization and of repolarization and increases the peak amplitude. Under voltage-clamp, dibutyryl cyclic AMP enhances the maximum ICa by increasing the conductance (ga), but without affecting its reversal potential (Ea) and kinetics in preparations in normal Krebs solution as well as in preparations in tetraethylammonium chloride loading solution. In normal Krebs solution, dibutyryl cyclic AMP also enhances the peak (Ib') and late outward K+ currents (Ib) by increasing the conductances (gb') and (gb), respectively. These results indicate that in vas deferens smooth muscle intracellular cyclic AMP may be of functional significance for activation of voltage-dependent peak and late IK channels as well as activation of voltage-dependent ICa channel.

Nitrinergic nerves controlling pacemaker activities of the inner sublayer (P-layer) in the canine proximal colon circular muscles.

The inner sublayer (P-layer) of the circular muscle coat in the canine proximal colon has been known to produce spontaneous mechanical contractions associated with characteristic electrical activities called slow waves. We recorded the mechanical activities of tissue preparations from this P-layer. Normal Krebs solution (K+; 6 mM) was used as the perfusate. Elevation of extracellular K+ concentrations in the range of 12 mM and 36 mM induced intensified phasic contractions. Administration of an NO-synthase inhibitor, N omega-nitro-arginine methyl ester (L-NAME, 50 microM), enhanced both the spontaneous mechanical rhythms and high extracellular K(+)-induced contractions. Administration of the substrate for NO synthases, L-arginine (400 microM) remarkably suppressed the effects of L-NAME on the amplitude of the spontaneous rhythms and on responses to extracellular high K+. Histological structures of nerves in the P-layer were investigated by an NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase technique and by the immunohistochemistry of NO-synthases, since NO-producing (nitrinergic) nerves usually, if not always, show a histochemical NADPH-diaphorase positive reaction in formaldehyde-fixed specimens, and since features of ganglia and nerve strands in the outer subdivision of the submucosal plexus (plexus submucosus externus; or so-called Henle's plexus) together with the delicate network of nerve terminal varicosities within the P-layer were clearly visualized by this method. The topographical arrangement of nitrinergic nerves supported the view that they produce nitric oxide (NO), being one of the major chemical mediators of the neural control of the spontaneous rhythms in the P-layer.

The role of capacitative Ca2+ influx in the alpha 1B-adrenoceptor-mediated contraction to phenylephrine of the rat spleen.

1. The mechanism of contraction to phenylephrine in the rat spleen (mediated via alpha 1B-adrenoceptors) has been studied in functional experiments. 2. The concentration-dependent contraction of the rat spleen to cumulative additions of phenylephrine (pD2 4.8 +/- 0.1) was not significantly reduced by the selective protein kinase C (PKC) inhibitor, calphostin C (10(-6)M) or potentiated by the DAG kinase inhibitor, R59022 (10(-6) M). 3. Contraction of the rat spleen in normal Krebs solution containing Ca2+ (2.5 mM) to a single concentration of phenylephrine (3 x 10(-4) M) produced a maximal response consisting of an initial phasic component and a more slowly developing tonic component. However in Ca(2+)-free Krebs solution (containing EGTA), phenylephrine (3 x 10(-4)M) produced only a phasic contraction which was reduced to 46 +/- 3% maximum response to phenylephrine in normal Krebs solution. 4. In some tissues after the contraction to phenylephrine (3 x 10(-4) M) in Ca(2+)-free Krebs solution (containing EGTA), the phenylephrine was washed out and the tissue was allowed to recover. After 2 h, upon addition of Ca2+ (2.5 mM) to the Krebs solution (EGTA now removed) a tonic contraction developed in the tissue (97 +/- 4% maximum response to phenylephrine). 5. Cyclopiazonic acid produced a tonic contraction of the rat spleen with a maximum effect at 10(-5) M (202 +/- 8% maximum response compared with that to phenylephrine). The contraction to CPA (10(-5) M) was reduced in Ca(2+)-free Krebs solution containing EGTA (30 +/- 4% of the maximum response to phenylephrine). One hour after the end of the contraction in Ca(2+)-free Krebs solution (EGTA now removed), upon addition of Ca2+ (2.5 mM) to the Krebs solution a tonic contraction developed in the tissue (263 +/- 12% maximum response to phenylephrine). 6 In Ca2+-free Krebs solution, after the spleen had been incubated with cyclopiazonic acid for 30 min,the subsequent contraction to phenylephrine (3 x 10-4 M) was reduced from 46+/-3% to 9+/-2%maximum response to phenylephrine.7 Cumulative contractions to phenylephrine and the contraction to cyclopiazonic acid (10-5 M) in the spleen were not significantly affected by nifedipine (10-6 M). The non-selective Ca2+channel blocker,SK&F 96365 (3 x 10-5 M) reduced the maximum response for the cumulative additions of phenylephrine to 35+/-1% and the contraction to CPA (10-5 M) from 202+/-8% to 108+/-8% maximum response to phenylephrine.8 The tyrosine kinase inhibitors genistein (3 x 10-5 M and tyrphostin 23 (10-4 M), reduced the maximum response to phenylephrine in the spleen to 51+/-4% and 44+/-5% respectively and the maximum contraction to cyclopiazonic acid (3 x 10-6 M) in the spleen from 132 +/- 6% to 82 +/-5% and 80 +/- 7% maximum response to phenylephrine respectively without affecting contractions to K+.9 In conclusion, these results are consistent with the contraction of the rat spleen to phenylephrine consisting of an initial phasic contraction due to release of intracellular Ca2+ and a larger tonic contraction due to capacitative Ca2+ influx through non-voltage-gated Ca2+ channels and which may involve a tyrosine kinase. This suggests that inositol triphosphate but not diacylglycerol is involved in the contraction.

Electrophysiological actions of phenytoin on N‐methyl‐D‐aspartate receptor‐mediated responses in rat hippocampus in vitro

1. The effects of the anticonvulsant, phenytoin, have been examined on N-methyl-D-aspartate (NMDA) receptor-mediated population spikes in the CA1 region of the rat hippocampus in vitro. 2. The 'conventional' (AMPA receptor-mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 x 10(-6) M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), after which superfusion with a nominally Mg(2+)-free Krebs solution (containing 5 x 10(-6) M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30-40 min. 3. The epileptiform population spike was abolished by the non-competitive NMDA antagonist, dizocilpine (1 x 10(-6) M, 20-30 min) and inhibited by the competitive NMDA receptor antagonist, D-CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 x 10(-7) M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex. 4. Phenytoin (0.1, 0.3 and 1 x 10(-4) M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor-mediated epileptiform population response (n = 7 slices). 5. Phenytoin (3 x 10-6 M to 1 x 10-4M) attenuated the effects of the sodium channel activator,veratridine (2 x 10-6 M), on the CAl population spike amplitude (recorded in normal Krebs solution),indicating that the previously observed lack of effect of phenytoin on the NMDA receptor-mediated response was not due to impaired access of phenytoin to the biophase.6. These data support the conclusion that antagonism of NMDA receptor-mediated events is not a pharmacological property of phenytoin and that such an action is therefore unlikely to contribute to the anticonvulsant activity of this drug.

Study of the in vivo and in vitro cardiovascular effects of (+)‐glaucine and N‐carbethoxysecoglaucine in rats

1. The cardiovascular and vasorelaxant effects of (+)-glaucine and of a semisynthetic derivative (N-carbethoxysecoglaucine) were studied in rats. 2. N-carbethoxysecoglaucine did not modify either systolic arterial pressure or heart rate values in conscious (25 mg kg-1, p.o.) and anaesthetized normotensive rats (5 mg kg-1, i.v.). Furthermore, this compound showed no activity in the experiments carried out on rat isolated aorta [contractility and 45Ca2+ influx assays (5 microM)] and did not modify the rate and force of contraction in rat isolated atria (5 microM). 3. In conscious normotensive rats, oral administration of (+)-glaucine (25 mg kg-1) did not modify either systolic arterial pressure or heart rate. 4. In anaesthetized normotensive rats, (+)-glaucine (5 mg kg-1, i.v.) produced a remarkable fall in mean arterial pressure (MAP) accompanied by a significant decrease in heart rate. In the same preparation, (+)-glaucine (5 mg kg-1, i.v.) did not modify the cardiovascular effects induced by noradrenaline (NA) (5 micrograms kg-1) and 5-hydroxytryptamine (5-HT) (300 micrograms kg-1) but markedly inhibited those induced by nicotine (200 micrograms kg-1). 5. In isolated intact aorta of rat, (+)-glaucine (0.15-5 microM) competitively inhibited the contractions induced by NA (with a pA2 value of 7.14) and non-competitively those induced by 5-HT (in normal Krebs solution) and Ca2+ (in depolarizing Ca(2+)-free high-K+ 50 mM solution), with depression of the maximal response and with pD2 values of 5.56 and 5.26, respectively. 6. In experiments in Ca2+-free medium, (+)-glaucine (3 microM) inhibited the contractions induced by NA and had no effect on either 5-HT- or caffeine-induced contractions.7. Furthermore, in the experiments with radioactive Ca2+, (+)-glaucine (3 microM) did not modify the basal uptake of 45Ca2+ but strongly inhibited the influx of 45Ca2+ induced by NA, 5-HT and K+.8. (+)-Glaucine (5microM) had no effect on rate and force of contraction in rat isolated atria.9. These results indicate that: (a) the cardiovascular effects (hypotension and bradycardia) of (+)-glaucine in anaesthetized normotensive rats (5 mg kg-1) may be due, at least in part, to a ganglioplexic effect; (b) the vasorelaxant action of ( + )-glaucine (0.15-5 microM) in rat isolated aorta can be attributed to an alpha1-adrenoceptor blocking property (which may explain its inhibition of noradrenaline-induced 45Ca2+influx and contractions in normal Krebs solution and noradrenaline-induced contractions in Ca2+-free medium) and to a Ca2+-antagonist activity (which may be responsible, at least in part, for the inhibition of 45Ca2+ uptake induced by NA, 5-HT and K+ and the contractions induced by both NA and 5-HT in normal Krebs solution and by Ca2+ in Ca2+-free high-K+ medium) and (c) there is no correlation between the mechanisms of action observed for (+ )-glaucine in vivo and in vitro, which suggests that the vasorelaxant activity of this alkaloid does not contribute to its hypotensive activity.

Effects of levcromakalim and glibenclamide on paced guinea-pig atrial strips exposed to hypoxia

Isolated strips of guinea-pig atrial myocardium were mounted in isometric myographs and electrically paced for measurements of myocardial contractile function. Levcromakalim, a K + channel opener, completely inhibited the contractile force in a concentration-dependent way (EC 50 = 15 μM). Glibenclamide (3 μM), a blocker of ATP-regulated K + channels (K ATP), caused a 5-fold rightward shift of the concentration-effect curve. Exposure of the atrial strips to hypoxia caused a time-dependent loss of contractility from 100% to a minimum level of 60% within 12 min. Levcromakalim (1 μM, 3 μM and 10 μM) concentration-dependently enhanced the hypoxia-induced inhibition of contractile function whereas levcromakalim (0.01 μM and 0.1 μM) had no significant effect. In the presence of levcromakalim (10 μM) hypoxia reduced the contractile force to 25%. Glibenclamide (3 μM) totally antagonized the enhancing effect of levcromakalim. When hypoxia was induced in glucose-free Krebs solution with 2-deoxyglucose, the myocardial contractility was completely suppressed within 12 min. Glibenclamide by itself (3 μM) failed to influence the myocardial response to hypoxia both in normal Krebs solution and under conditions of impaired glycolysis. The results indicate that levcromakalim by activation of myocardial ATP-regulated K + channels accelerates and enhances the hypoxia-induced inhibition of myocardial contractile function. This effect may possibly contribute to the mechanism by which K + channel openers exert cardioprotection. The results further suggest that mechanisms different from activation of K ATP take a major part in the depressant mechanical response to hypoxia and glycolytic blockade in the guinea-pig atrial myocardium.

Properties of intracellular calcium stores and their role in receptor-mediated catecholamine secretion in rat adrenal chromaffin cells.

Simulation of rat adrenal chromaffin cells either with receptor agonists such as muscarine, bradykinin and histamine or with caffeine in Ca(2+)-free Krebs solution induced a brief increase in the intracellular free Ca2+ concentration, [Ca2+]i, which terminated within 90 s. Both the agonist- and caffeine-induced [Ca2+]i responses were abolished in cells which had been treated with either 500 nM thapsigargin (TG) or 20 microM ryanodine, suggesting that inositol trisphosphate and caffeine cause Ca2+ release either from the same Ca2+ store or from distinct stores which exchange Ca2+ rapidly. In normal Krebs solution, these agonists evoked catecholamine (CA) secretion which showed an initial transient followed by a sustained component. Neither component of the secretion was significantly affected by TG or ryanodine. In the medium containing 16 microM Ca2+ or no Ca2+, CA secretion evoked by 30-second stimulation with 100 microM muscarine was 59 or 7%, respectively, of that evoked in the normal medium containing 2 mM Ca2+. In TG-treated cells, the CA secretion at 16 microM Ca2+ was reduced to 23% and that in the Ca(2+)-free medium was completely abolished. These results suggest that the receptor-mediated Ca2+ entry solely determines the rate of CA secretion in rat chromaffin cells when stimulated by receptor agonists in the normal medium, whereas intracellular Ca2+ release and Ca2+ entry may cooperatively support the secretion when cells are stimulated in media containing low concentrations of Ca2+.