(1/6807) Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes.
Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit. (+info)
(2/6807) [3H]-Mesulergine labels 5-HT7 sites in rat brain and guinea-pig ileum but not rat jejunum.
1. The primary aim of this investigation was to determine whether binding sites corresponding to the 5-HT7 receptor could be detected in smooth muscle of the rat jejunum. Binding studies in rat brain (whole brain minus cerebellum) and guinea-pig ileal longitudinal muscle were also undertaken in order to compare the binding characteristics of these tissues. Studies were performed using [3H]-mesulergine, as it has a high affinity for 5-HT7 receptors. 2. In the rat brain and guinea-pig ileum, pKD values for [3H]-mesulergine of 8.0 +/- 0.04 and 7.9 +/- 0.11 (n = 3) and Bmax values of 9.9 +/- 0.3 and 21.5 +/- 4.9 fmol mg(-1) protein were obtained respectively, but no binding was detected in the rat jejunum. [3H]-mesulergine binding in the rat brain and guinea-pig ileum was displaced with the agonists 5-carboxamidotryptamine (5-CT) > 5-hydroxytryptamine (5-HT) > or = 5-methoxytryptamine (5-MeOT) > sumatriptan and the antagonists risperidone > or = LSD > or = metergoline > ritanserin > > pindolol. 3. Despite the lack of [3H]-mesulergine binding in the rat jejunum, functional studies undertaken revealed a biphasic contractile response to 5-HT which was partly blocked by ondansetron (1 microM). The residual response was present in over 50% of tissues studied and was found to be inhibited by risperidone > LSD > metergoline > mesulergine = ritanserin > pindolol, but was unaffected by RS 102221 (3 microM), cinanserin (30 nM), yohimbine (0.1 microM) and GR 113808 (1 microM). In addition, the agonist order of potency was 5-CT > 5-HT > 5-MeOT > sumatriptan. 4. In conclusion, binding studies performed with [3H]-mesulergine were able to detect 5-HT7 sites in rat brain and guinea-pig ileum, but not in rat jejunum, where a functional 5-HT7-like receptor was present. (+info)
(3/6807) Effects of tumour necrosis factor-alpha on left ventricular function in the rat isolated perfused heart: possible mechanisms for a decline in cardiac function.
1. The cardiac depressant actions of TNF were investigated in the isolated perfused rat heart under constant flow (10 ml min(-1)) and constant pressure (70 mmHg) conditions, using a recirculating (50 ml) mode of perfusion. 2. Under constant flow conditions TNF (20 ng ml(-1)) caused an early (< 25 min) decrease in left ventricular developed pressure (LVDP), which was maintained for 90 min (LVDP after 90 min: control vs TNF; 110 +/- 4 vs 82 +/- 10 mmHg, P < 0.01). 3. The depression in cardiac function seen with TNF under constant flow conditions, was blocked by the ceramidase inhibitor N-oleoylethanolamine (NOE), 1 microM, (LVDP after 90 min: TNF vs TNF with NOE; 82 +/- 10 vs 11 +/- 5 mmHg, P < 0.05). 4. In hearts perfused at constant pressure, TNF caused a decrease in coronary flow rate (change in flow 20 min after TNF: control vs TNF; -3.0 +/- 0.9 vs -8.7 +/- 1.2 ml min(-1), P < 0.01). This was paralleled by a negative inotropic effect (change in LVDP 20 min after TNF: control vs TNF; -17 +/- 7 vs -46 +/- 6 mmHg, P < 0.01). The decline in function was more rapid and more severe than that seen under conditions of constant flow. 5. These data indicate that cardiac function can be disrupted by TNF on two levels, firstly via a direct, ceramidase dependant negative inotropic effect, and secondly via an indirect coronary vasoconstriction. (+info)
(4/6807) Modulation of chloride, potassium and bicarbonate transport by muscarinic receptors in a human adenocarcinoma cell line.
1. Short-circuit current (I(SC)) responses to carbachol (CCh) were investigated in Colony 1 epithelia, a subpopulation of the HCA-7 adenocarcinoma cell line. In Krebs-Henseleit (KH) buffer, CCh responses consisted of three I(SC) components: an unusual rapid decrease (the 10 s spike) followed by an upward spike at 30 s and a slower transient increase (the 2 min peak). This response was not potentiated by forskolin; rather, CCh inhibited cyclic AMP-stimulated I(SC). 2. In HCO3- free buffer, the decrease in forskolin-elevated I(SC) after CCh was reduced, although the interactions between CCh and forskolin remained at best additive rather than synergistic. When Cl- anions were replaced by gluconate, both Ca2+- and cyclic AMP-mediated electrogenic responses were significantly inhibited. 3. Basolateral Ba2+ (1-10 mM) and 293B (10 microM) selectively inhibited forskolin stimulation of I(SC), without altering the effects of CCh. Under Ba2+- or 293B-treated conditions, CCh responses were potentiated by pretreatment with forskolin. 4. Basolateral charybdotoxin (50 nM) significantly increased the size of the 10 s spike of CCh responses in both KH and HCO3- free medium, without affecting the 2 min peak. The enhanced 10 s spike was inhibited by prior addition of 5 mM apical Ba2+. Charybdotoxin did not affect forskolin responses. 5. In epithelial layers prestimulated with forskolin, the muscarinic antagonists atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, both at 100 nM) abolished subsequent 10 microM CCh responses. Following addition of p-fluoro hexahydro-sila-difenidol (pF-HHSiD, 10 microM) or pirenzepine (1 microM), qualitative changes in the CCh response time-profile also indicated a rightward shift of the agonist concentration-response curve; however, 1 microM gallamine had no effect. These results suggest that a single M3-like receptor subtype mediates the secretory response to CCh. 6. It is concluded that CCh and forskolin activate discrete populations of basolateral K+ channels gated by either Ca2+ or cyclic AMP, but that the Cl- permeability of the apical membrane may limit their combined effects on electrogenic Cl- secretion. In addition, CCh activates a Ba2+-sensitive apical K+ conductance leading to electrogenic K+ transport. Both agents may also modulate HCO3- secretion through a mechanism at least partially dependent on carbonic anhydrase. (+info)
(5/6807) Mechanisms involved in the metabotropic glutamate receptor-enhancement of NMDA-mediated motoneurone responses in frog spinal cord.
1. The metabotropic glutamate receptor (mGluR) agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10-100 microM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 microM). 2. In the presence of NMDA open channel blockers [Mg2+; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801); 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] and TTX, trans-ACPD significantly potentiated NMDA-induced motoneurone depolarizations, but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)- or kainate-induced depolarizations. 3. NMDA potentiation was blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (240 microM), but not by alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (MCCG) (290 microM) or by alpha-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 microM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 microM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 microM). Therefore, trans-ACPD's facilitatory effects appear to involve group I mGluRs. 4. Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3-6 ng ml(-1), 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 microM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCI (H9) (77 microM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 microM) had no effect. 5. Intracellular Ca2+ depletion with thapsigargin (0.1 microM) (which inhibits Ca2+/ATPase), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 microM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+-free medium all reduced trans-ACPD's effects. 6. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 microM) and chlorpromazine (100 microM) diminished the potentiation. 7. In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+-produced channel block of the NMDA receptor. (+info)
(6/6807) Treatment with amprenavir alone or amprenavir with zidovudine and lamivudine in adults with human immunodeficiency virus infection. AIDS Clinical Trials Group 347 Study Team.
Amprenavir is a human immunodeficiency virus (HIV) protease inhibitor with a favorable pharmacokinetic profile and good in vitro activity. Ninety-two lamivudine- and protease inhibitor-naive individuals with >/=50 CD4 cells/mm3 and >/=5000 HIV RNA copies/mL were assigned amprenavir (1200 mg) alone or with zidovudine (300 mg) plus lamivudine (150 mg), all given every 12 h. After a median follow-up of 88 days, the findings of a planned interim review resulted in termination of the amprenavir monotherapy arm. Among 85 subjects with confirmed plasma HIV RNA determination, 15 of 42 monotherapy versus 1 of 43 triple-therapy subjects had an HIV RNA increase above baseline or 1 log10 above nadir (P=.0001). For subjects taking triple therapy at 24 weeks, the median decrease in HIV RNA was 2.04 log10 copies/mL, and 17 (63%) of 27 evaluable subjects had <500 HIV RNA copies/mL. Treatment with amprenavir, zidovudine, and lamivudine together reduced the levels of HIV RNA significantly more than did amprenavir monotherapy. (+info)
(7/6807) Isoforms of the Na-K-2Cl cotransporter in murine TAL II. Functional characterization and activation by cAMP.
The functional properties of alternatively spliced isoforms of the mouse apical Na+-K+-2Cl- cotransporter (mBSC1) were examined, using expression in Xenopus oocytes and measurement of 22Na+ or 86Rb+ uptake. A total of six isoforms, generated by the combinatorial association of three 5' exon cassettes (A, B, and F) with two alternative 3' ends, are expressed in mouse thick ascending limb (TAL) [see companion article, D. B. Mount, A. Baekgaard, A. E. Hall, C. Plata, J. Xu, D. R. Beier, G. Gamba, and S. C. Hebert. Am. J. Physiol. 276 (Renal Physiol. 45): F347-F358, 1999]. The two 3' ends predict COOH-terminal cytoplasmic domains of 129 amino acids (the C4 COOH terminus) and 457 amino acids (the C9 terminus). The three C9 isoforms (mBSC1-A9/F9/B9) all express Na+-K+-2Cl- cotransport activity, whereas C4 isoforms are nonfunctional in Xenopus oocytes. Activation or inhibition of protein kinase A (PKA) does not affect the activity of the C9 isoforms. The coinjection of mBSC1-A4 with mBSC1-F9 reduces tracer uptake, compared with mBSC1-F9 alone, an effect of C4 isoforms that is partially reversed by the addition of cAMP-IBMX to the uptake medium. The inhibitory effect of C4 isoforms is a dose-dependent function of the alternatively spliced COOH terminus. Isoforms with a C4 COOH terminus thus exert a dominant negative effect on Na+-K+-2Cl- cotransport, a property that is reversed by the activation of PKA. This interaction between coexpressed COOH-terminal isoforms of mBSC1 may account for the regulation of Na+-K+-2Cl- cotransport in the mouse TAL by hormones that generate cAMP. (+info)
(8/6807) Effect of 5-HT4 receptor stimulation on the pacemaker current I(f) in human isolated atrial myocytes.
OBJECTIVE: 5-HT4 receptors are present in human atrial cells and their stimulation has been implicated in the genesis of atrial arrhythmias including atrial fibrillation. An I(f)-like current has been recorded in human atrial myocytes, where it is modulated by beta-adrenergic stimulation. In the present study, we investigated the effect of serotonin (5-hydroxytryptamine, 5-HT) on I(f) electrophysiological properties, in order to get an insight into the possible contribution of I(f) to the arrhythmogenic action of 5-HT in human atria. METHODS: Human atrial myocytes were isolated by enzymatic digestion from samples of atrial appendage of patients undergoing coeffective cardiac surgery. Patch-clamped cells were superfused with a modified Tyrode's solution in order to amplify I(f) and reduce overlapping currents. RESULTS AND CONCLUSIONS: A time-dependent, cesium-sensitive increasing inward current, that we had previously described having the electrophysiological properties of the pacemaker current I(f), was elicited by negative steps (-60 to -130 mV) from a holding potential of -40 mV. Boltzmann fit of control activation curves gave a midpoint (V1/2) of -88.9 +/- 2.6 mV (n = 14). 5-HT (1 microM) consistently caused a positive shift of V1/2 of 11.0 +/- 2.0 mV (n = 8, p < 0.001) of the activation curve toward less negative potentials, thus increasing the amount of current activated by clamp steps near the physiological maximum diastolic potential of these cells. The effect was dose-dependent, the EC50 being 0.14 microM. Maximum current amplitude was not changed by 5-HT. 5-HT did not increase I(f) amplitude when the current was maximally activated by cAMP perfused into the cell. The selective 5-HT4 antagonists, DAU 6285 (10 microM) and GR 125487 (1 microM), completely prevented the effect of 5-HT on I(f). The shift of V1/2 caused by 1 microM 5-HT in the presence of DAU 6285 or GR 125487 was 0.3 +/- 1 mV (n = 6) and 1.0 +/- 0.6 mV (n = 5), respectively (p < 0.01 versus 5-HT alone). The effect of 5-HT4 receptor blockade was specific, since neither DAU 6285 nor GR 125487 prevented the effect of 1 microM isoprenaline on I(f). Thus, 5-HT4 stimulation increases I(f) in human atrial myocytes; this effect may contribute to the arrhythmogenic action of 5-HT in human atrium. (+info)