microM-100 microM Research Articles

Prostaglandin F<sub>2</sub><sub>α</sub> Regulates the Nitric Oxide Generating System in Bovine Luteal Endothelial Cells

The objective of the present study was to elucidate whether luteolytic prostaglandin F(2alpha) (PGF) plays roles in regulating the nitric oxide (NO) generating system in luteal endothelial cells (LECs). Reverse transcriptase PCR, immunoblotting and immunostaining revealed the presence of PGF receptor mRNA (521 bp) and protein (64 kDa) in cultured LECs obtained from the mid-stage corpus luteum. When cultured LECs were exposed to 0.1 microM-10 microM PGF, NO production was significantly stimulated by PGF at 24 h. When LECs were exposed to 1 microM PGF for 2, 6 and 24 h, PGF did not affect the expressions of endothelial NO synthase (eNOS) mRNA and protein. On the other hand, PGF stimulated the expression of inducible NOS (iNOS) mRNA (P<0.05) and protein (P<0.05) at 2 h, but not at 6 and 24 h. By observing the conversion of [(3)C](L)-arginine to [(3)C](L)-citrulline, we found that PGF stimulated NOS activity in cultured LECs at 2 h (P<0.05). The overall findings indicate that bovine LECs are a target for PGF and that PGF stimulates iNOS expression and NOS activity in bovine LECs. Stimulation of the NO generating system and NOS activity by PGF may result in increasing local NO production followed by luteolysis.

Large conductance Ca2+-activated K+ (BK) channel: Activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.

Extracellular ATP activates chloride and taurine conductances in cultured hippocampal neurons.

We investigated regulation by extracellular ATP of channels important for volume regulation of rat hippocampal neurons. Cultures made from fetuses at the eighteenth gestational day were predominantly neuronal after 10-20 days in vitro, as indicated by immunostaining for neuron specific enolase. Neurons recorded with whole-cell patch clamp showed inward currents when membrane voltages were driven to values greater than -50 mV. Chloride conductance increased with 10 microM-100 microM extracellular ATP in a dose-dependent fashion. Similarly, an increase in taurine conductance was observed with 50 microM ATP. These currents were inhibited by the anion channel and purinergic receptor antagonists niflumic acid and suramin, respectively. The chloride conductance response to 10 microM ATP was increased over eight-fold in hypoosmotic medium (250 mOsm); however, chloride conductance in 0 mM ATP was not altered by this osmolality. Thus anion and osmolyte conducting channels activated via purinergic receptors may mediate volume regulation of hippocampal neurons.

Existence of functional M3-muscarinic receptors in the human heart.

It has been recently shown that, in adult rat ventricular cardiomyocytes, functional muscarinic receptors (M-receptors) of the M(3)-subtype exist that mediate inositol phosphate (IP) formation. The aim of this study was to characterize the M-receptor subtype mediating IP formation in the human heart. For this purpose in [3H]-myo-inositol labeled slices of human right atria, carbachol-induced [3H]-IP formation and its inhibition by several M-receptor antagonists was assessed. Carbachol (0.1 microM-100 microM) increased [3H]-IP formation; maximal increase at 100 microM was 93+/-16% above basal ( n=20); the pEC(50)-value for carbachol was 5.56. Atropine (1 microM) completely suppressed 100 microM carbachol-induced [3H]-IP formation. Among the M-receptor subtype "selective" antagonists himbacine (1 microM) and pirenzepine (1 microM) only marginally affected carbachol-induced [3H]-IP formation whereas the M(3)-receptor antagonist darifenacin (1 nM-1 microM) concentration-dependently inhibited carbachol-induced [3H]-IP formation with a pK(i)-value of 8.49. We conclude that in human right atrium there exist functional M(3)-receptors that couple to IP formation.

Real-time direct measurement of nitric oxide in bovine perfused eye trabecular meshwork using a clark-type electrode.

NO was detected in bovine trabecular meshwork (TM). Bovine eyes were perfused (posterior ciliary artery). In some eyes (operated eyes) a NO electrode was inserted adjacent to the TM (scleral flap). Vascular perfusion/intraocular pressures (VPP/IOP) were recorded. In operated eyes, epinephrine (1 nM-100 microM) increased NO (maximally 979.9 +/- 117.6 nM, mean +/- SDM). Timolol (1 mM) shifted the epinephrine-NO concentration-response curve rightward (2.94 log units) without significantly changing the maximal response (903.0 +/- 67.7 nM, mean +/- SDM). The non-selective NO synthase (NOS) inhibitor L-NMMA (100 microM) virtually abolished the NO response to epinephrine. L-NMMA alone (1 microM-100 microM) significantly reduced tonic NO generation (maximally 109.5 +/- 24.9 nM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. In unoperated eyes, epinephrine (1 nM-100 microM) reduced IOP (maximally 2.56 +/- 0.64 mmHg, mean +/- SDM). Epinephrine (100 microM) mildly increased VPP (4.6 +/- 1.3 mmHg, mean +/- SDM). Baseline aqueous humor formation rate (11.5 +/- 3.2 microl/min, mean +/- SDM) was unaffected. Effluent perfusate (effusate) total NO(2)(-) was determined by enzymatically reducing all NO(3)(-) to NO(2)(-), then assessing resultant NO(2)(-) (Griess assay). Epinephrine (1 nM-1 microM) increased effusate NO(2)(-) (maximally 15.8 +/- 4.9 microM, mean +/- SDM). Timolol (1 mM) reduced, and L-NMMA (100 microM) virtually abolished effusate NO(2)(-) response to epinephrine. L-NMMA alone (1 microM-100 microM) reduced tonic effusate NO(2)(-) (maximally from 5.8 +/- 1.6 microM to 1.1 +/- 0.9 microM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. NO is generated tonically in bovine TM and increases in response to epinephrine.

AMPA-preferring receptors mediate excitatory non-NMDA responses of primate retinal ganglion cells.

Glutamate and kainate-induced currents of primate ganglion cells were studied using the whole-cell patch-clamp technique in a retinal slice preparation. Antagonists and allosteric modulators of desensitization selective for either alpha-amino-3-hydroxy-5-methyl-4-isoazoleprionic acid (AMPA)- or kainate-preferring receptors were used to determine the contributions of each type of receptor to excitatory responses. With synaptic transmission and NMDA receptors blocked, the AMPA-preferring receptor antagonist GYKI 52466 (30 microM-100 microM) reversibly blocked most of the glutamate-induced current in ganglion cells. GYKI 52466 also blocked the response in ganglion cells to focally applied kainate, suggesting that the current response to kainate arises from activation of AMPA-preferring receptors, and not kainate-preferring receptors. Both cyclothiazide (10 microM-100 microM) and the novel drug 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide (PEPA, 20 microM-100 microM), which selectively enhance responses mediated by AMPA-preferring receptors, enhanced glutamate-induced responses of ganglion cells. Since these drugs preferentially inhibit desensitization of the flip and flop splice variants, respectively, of AMPA-preferring receptors, it is likely that both splice variants are present on these ganglion cells. Concanavalin A, which selectively suppresses the desensitization of kainate-preferring receptors, had no effect on the glutamate-induced responses of ganglion cells. We conclude that the non-NMDA component of the excitatory, glutamatergic input to primate ganglion cells is mediated largely by AMPA-preferring receptors, with little, if any, kainate-preferring receptor-mediated response.

A role for src tyrosine kinase in regulating adrenal aldosterone production.

Adrenal aldosterone synthesis is influenced by a variety of factors. The major physiological regulators of aldosterone production are angiotensin II (Ang IotaIota) and potassium (K(+)). Ang IotaIota stimulates aldosterone production through the activation of multiple intracellular signaling pathways. It has recently been demonstrated that Ang IotaIota activates src tyrosine kinases in vascular smooth muscle cells. The src family of tyrosine kinases are widely distributed non-receptor kinases that influence several signal transduction pathways. In the present study we evaluated the effect of a selective src family inhibitor, PP2, on aldosterone production using a human adrenocortical carcinoma-derived (H295R) cell line. Treatments for 6 or 48 h with PP2 (0.3 microM-10 microM) inhibited basal, Ang IotaIota, K(+) and dibutyryladenosine cyclic monophosphate (dbcAMP) stimulation of aldosterone production in a concentration-dependent manner. PP2 did not affect cell viability at any of the concentrations tested. Moreover, time course studies using PP2 (10 microM) for 6, 12, 24, and 48 h revealed a time-dependent inhibition of aldosterone production. Inhibition by PP2 (0.3-10 microM) was also observed for the metabolism of 22R-hydroxycholesterol (22R-OHChol) to aldosterone in H295R cells. Since 22R-OHChol is a substrate for cytochrome P450 side-chain cleavage enzyme (CYP11A) that does not require steroidogenic acute regulatory (StAR) protein for transport to the inner mitochondrial membrane, these results suggest that PP2 inhibition occurred beyond the rate-limiting step in aldosterone synthesis. Genistein, a non-specific tyrosine kinase inhibitor also blocked aldosterone production, but the inhibition was the result of a non-specific effect on 3beta-hydroxysteroid dehydrogenase (3betaHSD). In contrast, PP2 did not appear to act as a direct inhibitor of 3betaHSD activity. To further investigate the site of PP2 action, we examined its effect on H295R cell metabolism of [(14)C]progesterone using thin layer chromatography. PP2 treatment for 48 h caused an increase in the conversion of progesterone to 17alpha-hydroxyprogesterone. To determine if this apparent increase in 17alpha-hydroxylase activity was due to increased transcript, we examined the effect of PP2 on CYP17 mRNA. PP2 treatment caused an increase in CYP17 mRNA without an effect on 3betaHSD mRNA levels. Inhibition of protein synthesis with cycloheximide increased basal levels of CYP17 mRNA levels and blocked the induction observed by PP2. This suggests that new protein synthesis is a necessary part of PP2 induction of CYP17. Taken together these data suggest that the src tyrosine kinase inhibitor, PP2, is a potent inhibitor of aldosterone production. One mechanism for the inhibition is through an induction of CYP17 mRNA and enzyme activity. Src tyrosine kinases, therefore, may be involved with the promotion of a glomerulosa phenotype through the inhibition of CYP17 expression.

Modulation of CPP32 activity and induction of apoptosis in Human CEM X 174 lymphocytes by heptachlor, a chlorinated hydrocarbon insecticide.

Heptachlor is an organochlorine insecticide used worldwide for the control of pests both agriculturally and domestically. Its lipophilic structure allows it to bioaccumulate and pass through the food chain, exposing those who come in contact with it to its tumor promoting and possible carcinogenic effects. As a mechanism of tumor promotion, we explored the possibility of heptachlor suppressing the apoptotic process in human CEM x 174 lymphocytes. In this article, we describe the effect of heptachlor on the activity of the apoptosis protease CPP32. We show that heptachlor by itself was able to stimulate CPP32 activity at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was observed. Low concentrations of heptachlor (5 microM-10 microM) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80 microM-120 microM) augmented it. We also showed that heptachlor alone at relatively high concentrations induced apoptosis-associated changes in CEM x 174 cells including high molecular weight (HMW) DNA cleavage and chromatin condensation. From these results, it appears that heptachlor has tumor promoting-like effects at lower concentrations, and at higher concentrations induces apoptosis as a mechanism of cytotoxicity.

Possible involvement of K+ channel opening to the interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

We have previously shown that interleukin-1 beta relaxes vascular smooth muscle by the NO-dependent and independent mechanisms (Takizawa et al.: Eur. J. Pharmacol. 330: 143-150, 1997). In this study, we investigated the mechanism of NO-independent relaxation. Treatment of the rat aorta with interleukin-1 beta for 24 hr inhibited the high-K+ induced contraction by decreasing cytosolic Ca2+ level ([Ca2+]i). The relationship between [Ca2+]i and tension in intact muscle and the pCa-tension curves in permeabilized muscle suggested that Ca2+ sensitivity of contractile element was not changed after the interleukin-1 beta-treatment. After a treatment with interleukin-1 beta for 24 hr, contractile effects of phenylephrine (1 microM-10 microM) were markedly inhibited in the presence of L-NMMA (100 microM) applied to inhibit NO synthesis. A blocker of ATP-sensitive K+ channel, glibenclamide (1 microM), partially recovered the interleukin-1 beta-induced inhibition. In contrast, a blocker of Ca(2+)-activated K+ channel, charybdotoxin (0.1 microM), was ineffective. These results suggest that membrane hyperpolarization due to activation of ATP-sensitive K+ channels may partly be responsible for the NO-independent mechanism of interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

Functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type 4 inhibitor.

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.

Somatostatin sst2 receptor-mediated inhibition of parietal cell function in rat isolated gastric mucosa.

1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself.

The activation of P1‐ and P2‐purinoceptors in the guinea‐pig left atrium by diadenosine polyphosphates

1. The effects of P1, P2-di(adenosine) pyrophosphate (AP2A), P1, P3-di(adenosine) triphosphate (AP3A), P1,P4-di(adenosine) tetraphosphate (AP4A), P1,P5-di(adenosine) pentaphosphate (AP5A), ATP, alpha, beta-methylene ADP and 2-chloroadenosine (2-ClAd) were examined in the guinea-pig driven left atrium. 2. All these purine compounds except alpha, beta-methylene ADP produced a negative inotropic response with a rank order of potency of: 2-ClAd > > AP2A > or = ATP > or = AP4A = AP3A = AP5A. The EC50 value for 2-ClAd was approximately 1 microM, while those for the remaining compounds were in the range 10 microM-100 microM, alpha, beta-Methylene ADP (10-300 microM), a selective P2Y-purinoceptor agonist, produced a small positive inotropism. 3. The P1-purinoceptor antagonist, 8-para-sulphophenyltheophylline (8-pSPT, 20 microM) caused a right-ward shift in the concentration-response curves for 2-ClAd, ATP and AP2A, but converted the responses of AP3A, AP4A, and AP5A into positive inotropisms. 4. The non-selective P2-purinoceptor antagonist, suramin (300 microM), had no significant effect on the concentration-response curves for 2-ClAd, ATP or AP2A, but significantly antagonized inhibitory responses to AP3A, AP4A and AP5A, and excitatory responses to alpha, beta-methylene ADP. 5. In the presence of 8-pSPT (20 microM), suramin (300 microM) abolished the positive inotropic responses evoked by the dinucleotides. 6. ATP was degraded far more rapidly than any of the dinucleotides, and AP3A was the least stable of the diadenosine compounds. The relative order of stability was AP2A > AP4A = AP5A > AP3A > > ATP. Suramin (300 microM) reduced the rate of degradation of ATP and AP3A by approximately 30%. Suramin had no significant effect on the degradation of AP2A, AP4A or AP5A. 7. It is concluded that the diadenosine polyphosphates cause negative inotropic responses via P1-purinoceptors and a hitherto undefined suramin-sensitive P2-purinoceptor, and that they appear to have positive inotropic effects mediated via another suramin-sensitive P2-purinoceptor.

Stimulation of sodium pump by vasoactive intestinal peptide in guinea-pig isolated trachea: potential contribution to mechanisms underlying relaxation of smooth muscle.

1. Relaxation of airway smooth muscle induced by vasoactive intestinal peptide (VIP) is mediated by adenosine 3':5' cyclic monophosphate (cyclic AMP). An interaction between the synthesis of cyclic AMP and enzymic activity of the plasmalemmal sodium pump (Na(+)-K(+)-ATPase) exists in certain isolated cell systems. This study sought to determine the contribution of Na(+)-K(+)-ATPase activity to relaxation of airway smooth muscle evoked by VIP. 2. All experiments were performed on isolated strips of guinea-pig trachea from which the epithelium had been removed. VIP was a more potent relaxant in tissues that were contracted with carbachol than those contracted with an equi-effective depolarizing concentration of K+. 3. Ouabain (0.1 microM-10 microM) induced contraction of tracheal strips. Contraction to ouabain (5 microM) was abolished following incubation of tissues with K(+)-free, or Ca(2+)-free (+ EGTA, 0.1 mM) physiological solutions. The contractile response to ouabain (5 microM) was not influenced significantly by exposure of the tissues to atropine (1 microM), phentolamine (5 microM) and diphenhydramine (1 microM) for 60 min. 4. Tissues were incubated with ouabain (5 microM; 60 min) or K(+)-free physiological solution (60 min) to inhibit Na(+)-K(+)-ATPase activity. These procedures reduced relaxation induced by VIP, peptide histidine isoleucine, forskolin, isoprenaline and sodium nitroprusside. 5. Relaxation to VIP was impaired significantly following exposure of tissues to a low Na+ solution (30 min) or amiloride (500 microM; 30 min). 6. Ouabain-sensitive uptake of 86Rb was measured in tracheal strips (devoid of epithelium and cartilage) as an index of Na(+)-K(+)-ATPase activity. VIP (1 microM; 2 min) caused a 4.7 fold stimulation of ouabain-sensitive uptake of 86Rb. This effect was impaired significantly by low Na+ solution. 7. The results suggest that (i) relaxation of tracheal smooth muscle to VIP is sensitive to procedures that inhibit activity of Na(+)-K(+)-ATPase and invoke a role for altered sodium pump function in the mechanisms that underlie cyclic AMP-dependent relaxation; and (ii) VIP stimulates ouabain-sensitive uptake of 86Rb in airway smooth muscle in a Na(+)-dependent manner.

Effect of Terfenadine and KW-4679, a Novel Antiallergic Compound, on Action Potential of Guinea Pig Ventricular Myocytes

It has been reported that terfenadine caused torsade de pointes ventricular arrhythmias. The prolongation of action potential duration (APD) in ventricles is considered to be one of the mechanisms of this adverse effect. We examined the effect of antiallergic drugs, terfenadine and KW-4679 ((Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b, e]oxepin- 2-acetic acid hydrochloride), on action potentials in isolated guinea pig ventricular myocytes. Terfenadine (30 nM-1 microM) increased APD in a concentration-dependent manner. On the other hand, KW-4679 (0.1 microM-100 microM) exerted no significant effects on action potential parameters. These results present no evidence that KW-4679 has the possibility to cause ventricular arrhythmias.

Oestradiol-induced relaxation of rabbit basilar artery by inhibition of voltage-dependent Ca channels through GTP-binding protein.

1. Effects of oestradiol on the electrical and mechanical properties of the rabbit basilar artery were investigated by use of microelectrode, patch-clamp and isometric tension recording methods. 2. Oestradiol (10 nM-100 microM) relaxed arterial tissue pre-contracted by excess [K]o solution (30 mM) in a concentration-dependent manner. In Ca-free solution, histamine (10 microM) and caffeine (20 mM) each produced a phasic contraction, but oestradiol (10 microM) did not significantly affect their amplitude. 3. Oestradiol (< or = 100 microM) did not change the resting membrane potential of the artery whether in the presence or absence of TEA (10 mM). Action potentials observed in the presence of 10 mM TEA were abolished by oestradiol (100 microM). 4. Oestradiol (1 microM-100 microM) inhibited the voltage-dependent Ba current in a concentration-dependent manner. Oestradiol (100 microM) inhibited the Ba current observed in the presence of nicardipine (1 microM) more than that in the absence of nicardipine (to 31.0% vs 62.0% of control). 5. GTP gamma S (30 microM) in the pipette enhanced the inhibitory actions of oestradiol on the Ba current. On the other hand, with GDP beta S (1 mM) in the pipette, oestradiol failed to inhibit the Ba current. Pertussis toxin (PTX 3 micrograms ml-1) in the pipette totally prevented the inhibitory action of oestradiol on the Ba current. 6. Oestradiol (< or = 100 microM) had no significant effect on the outward K currents evoked by a membrane depolarization. 7. These results strongly suggest that oestradiol relaxes arterial tissue by inhibition of voltage-dependent Ca channels and that it inhibits both nicardipine-sensitive and -resistant Ca currents via a PTX-sensitive GTP-binding protein. The main target of oestradiol among the arterial Ca channels seems to be the nicardipine-resistant Ca channel, rather than the nicardipine-sensitive one.

Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.

1. SCA40 (1nM-10 microM), isoprenaline (1-300 nM) and levcromakalim (100 nM-10 microM) each produced concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachea. Propranolol (1 microM) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 microM) or 8-(p)-sulphophenyltheophylline (8-SPT; 140 microM). 2. An isosmolar, K(+)-rich (80 mM) Krebs solution increased tracheal tone, antagonized SCA40 (approximately 60 fold), antagonized isoprenaline (approximately 20 fold) and very profoundly depressed the log concentration-effect curve for levcromakalim. Nifedipine (1 microM) did not itself modify the relaxant actions of SCA40, isoprenaline or levcromakalim. However, nifedipine prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by the K(+)-rich medium. In contrast, nifedipine did not prevent the equivalent antagonism of levcromakalim. 3. Charybdotoxin (100 nM) increased tracheal tone, antagonized SCA40 (approximately 4 fold) and antagonized isoprenaline (approximately 3 fold). Nifedipine (1 microM) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4. Quinine (30 microM) caused little or no change in tissue tone and did not modify the relaxant action of isoprenaline. However, quinine antagonized SCA40 (approximately 2 fold). Nifedipine (1 microM) prevented the antagonism of SCA40 induced by quinine. 5. Tested on spontaneously-beating guinea-pig isolated atria SCA40 (1 nM-10 microM) increased the rate of beating in a concentration-dependent manner. Over the concentration-range 1 microM-10 microM, SCA40 also caused an increase in the force of atrial contraction. 6. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of SCA40 (1 micro M) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 micro M, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization.7. SCA40 (10 nM- 100 micro M) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE)than that of cyclic GMP PDE derived from homogenates of guinea-pig trachealis. Theophylline(1 micro M- 1O mM) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis.8. Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II.9. It is concluded that the tracheal relaxant action of SCA40 (1 nM- 1 micro M) does not involve the activation of beta-adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKca channels with consequent cellular hyperpolarization and voltage-dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 micro M) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 micro M, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKca channels.

Postnatal changes in N-methyl-D-aspartate receptor binding and stimulation by glutamate and glycine of [3H]-MK-801 binding in human temporal cortex.

1. Homogenates of human infant and adult temporal cortex were used to measure [3H]-TCP and [3H]-MK-801 binding to the N-methyl-D-aspartate (NMDA)-coupled ion channel phencyclidine site. 2. Both [3H]-TCP and [3H]-MK-801 binding increased in infant cortex by > 100% between term and 26 weeks suggesting that the numbers of NMDA receptors increase during postnatal brain development. 3. [3H]-MK-801 binding was measured under non-equilibrium conditions in temporal cortex homogenates with the addition of 100 microM of L-glutamate plus a range of concentrations (0.05 microM-100 microM) of glycine. Glutamate and glycine increased [3H]-MK-801 binding by stimulating NMDA receptors and improving [3H]-MK-801 access to ion channel binding sites; maximum stimulation in adult and infant temporal cortex was achieved with 100 microM glutamate plus 5 microM glycine; a higher concentration of glycine (50 microM) reduced [3H]-MK-801 binding to below maximum. 4. The stimulation by 100 microM glutamate plus 5 microM glycine of [3H]-MK-801 binding in infant temporal cortex was affected by postnatal age. For example, although the stimulation of [3H]-MK-801 binding in 5-6 week infant cortex (236% of basal) was similar to adult cortex (230% of basal), in samples taken from infants aged 5-6 months glycine (plus glutamate) stimulation of [3H]-MK-801 binding (392% of basal) was substantially greater than that measured in adult temporal cortex. 5. The binding of [3H]-glycine to the glycine modulatory site associated with the NMDA receptor in infant cortex also increased with postnatal age by > 100% between term and 26 weeks. 6. It is concluded that NMDA receptors in infant cortex increase to levels greater than those in adult cortex during postnatal development. The results do not exclude the possibility that the transiently increased NMDA receptor-ion channel complex in infant cortex shows enhanced responses to agonists and modulators.

Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or skeletal SR Ca2+-release channel activity.6. At concentrations up to 1 microM, digoxin had no effect on Ca2+-induced 45Ca2+ efflux from skeletal muscle SR vesicles nor did it affect skeletal SR Ca2+-release channel Po, reflecting a difference between the cardiac and skeletal isoforms of the Ca2+-release channel.7. Since activation of the cardiac SR Ca2+-release channel occurs within the range of concentrations of digoxin encountered therapeutically, it is possible that activation of this channel contributes to the positive inotropic effect observed with this drug. Further, activation of the channel by higher concentrations of digoxin may contribute to the toxic effects seen clinically.

Endothelium-dependent modulation of resistance vessel contraction: studies with NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine.

1. The effect of NG-nitro-L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NOARG) on noradrenaline (NA)-induced contractility and acetylcholine (ACh)-induced endothelium-dependent relaxation was studied in rat mesenteric resistance arteries. 2. Third order branches of mesenteric arteries were dissected and mounted on two forty micron wires in a Mulvany myograph. 3. Incubation with L-NAME and L-NOARG (10 microM) caused a time-dependent shift in the 50% response to NA (ED50) (0.01 microM-10 microM) but was not associated with an increase in the maximum contractile response. 4. L-NAME and L-NOARG (10 microM) caused a time-dependent inhibition of ACh (1 microM)-induced relaxation with a maximum effect after 120 min. 5. Following endothelium removal, incubation with either L-NAME or L-NOARG caused no significant shift in the ED50, although the residual relaxation response to ACh (1 microM) was further attenuated. 6. Incubation with the cyclo-oxygenase inhibitor, indomethacin, enhanced the relaxation to ACh and reduced the inhibitory effects of L-NAME and L-NOARG. 7. In conclusion, L-NAME and L-NOARG are potent inhibitors of acetylcholine-induced endothelium-dependent relaxation in mesenteric resistance arteries. The shift in ED50 associated with these inhibitors suggests a probable role for the endothelium in modulating the contractility of the resistance vasculature.

Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed BK3 receptors in the guinea-pig trachea, and are blocked by HOE 140.

1. Bradykinin (BK) receptors of the guinea-pig taenia caeci were compared with those of the guinea-pig trachea, a preparation proposed to possess novel BK3 receptors. 2. Bradykinin-evoked contractile responses were unaffected in both preparations by the selective BK1 receptor antagonist [des-Arg9,Leu8]-BK (1 microM-10 microM). The BK2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-BK and D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK, both had low affinities (apparent pKB estimates less than 6) which did not differ significantly between the two preparations (P greater than 0.05). In contrast, the novel bradykinin receptor antagonist D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140) potently antagonized responses to bradykinin with relatively high affinity (apparent pKB = 8.42 +/- 0.15 and 8.94 +/- 0.16 in the taenia caeci, and trachea, respectively). 3. We conclude that the bradykinin receptors in the guinea-pig taenia caeci have similar recognition properties to those present in the guinea-pig trachea, and in this respect the taenia caeci represents a useful preparation for the further study of proposed novel BK3 receptors.