Photolytic manipulation of [Ca2+]i reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hippocampal pyramidal neurons. (1/91)

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sIAHP) remains unknown. We studied sIAHP in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+]i) peaked earlier and decayed more rapidly than sIAHP. Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sIAHP. In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+]i was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 msec), whereas the apamin-insensitive current activated more slowly (180 msec). The apamin-insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sIAHP channels. When [Ca2+]i was decreased rapidly via photolysis of diazo-2, the decay of sIAHP was similar to control (1. 7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sIAHP have intrinsically slow kinetics because of their high affinity for calcium.  (+info)

Novel action of BAPTA series chelators on intrinsic K+ currents in rat hippocampal neurones. (2/91)

1. Whole-cell recordings were made from rat CA1 neurones in brain slices. When electrodes contained diazo-2 (2 mM) or dibromo BAPTA (1 mM) a large steady-state outward current (hundreds of picoamps) developed within 5 min of breakthrough at a VH of -60 mV. BAPTA itself (1 mM) caused qualitatively similar but smaller effects. 2. The outward current was accompanied by increased conductance with a null potential close to the calculated K+ equilibrium potential (EK) of -110 mV. Development of outward current occurred concurrently with progressive loss of slow AHP tail current (IsAHP) evoked by brief depolarizations. The peak latency of IsAHP increased during the onset of chelator action. 3. The persistent outward current was reversibly inhibited by noradrenaline (10 microM) or isoprenaline (2-5 microM), and completely prevented by 8-bromoadenosine 3',5' cyclic monophosphate (8-Br cAMP; 100 microM) or QX-314 (10 mM) in recording electrodes. After development of outward current, diazo-2 photolysis caused inward current and decreased conductance. Both flash- and noradrenergic-sensitive responses were inwardly rectifying outward currents with null potentials close to EK. 4. The outward current induced by dibromo BAPTA was not blocked by internal EGTA (10 mM). However, experiments incorporating Ca2+ influx or Ca2+ loading of the buffer indicate that Ca2+ facilitated the outward current. 5. The outward currents induced by dibromo BAPTA or diazo-2 were not associated with significant changes in resting [Ca2+]i. Regions of the cell contributing to the outward current were deduced from measurements of fura-2 diffusion. These were compared with regions of [Ca2+]i elevation during IsAHP. 6. These results are consistent with the hypothesis that the BAPTA series Ca2+ buffers can activate those Ca2+-activated K+ channels that underlie the slow AHP, without the predicted elevation of bulk [Ca2+]i. Therefore these results cannot be interpreted solely in terms of Ca2+ concentration changes, although the observations illustrate a novel, investigative role for these compounds in the study of Ca2+-dependent processes.  (+info)

Non-prostanoid prostacyclin mimetics as neuronal stimulants in the rat: comparison of vagus nerve and NANC innervation of the colon. (3/91)

The spontaneous activity of the rat isolated colon is suppressed by prostacyclin analogues such as cicaprost (IC(50)=4.0 nM). Activation of prostanoid IP(1)-receptors located on NANC inhibitory neurones is involved. However, several non-prostanoids, which show medium to high IP(1) agonist potency on platelet and vascular preparations, exhibit very weak inhibitory activity on the colon. The aim of the study was to investigate this discrepancy. Firstly, we have demonstrated the very high depolarizing potency of cicaprost on the rat isolated vagus nerve (EC(50)=0.23 nM). Iloprost, taprostene and carbacyclin were 7.9, 66, and 81 fold less potent than cicaprost, indicating the presence of IP(1) as opposed to IP(2)-receptors. Three non-prostanoid prostacyclin mimetics, BMY 45778, BMY 42393 and ONO-1301, although much less potent than cicaprost (195, 990 and 1660 fold respectively), behaved as full agonists on the vagus nerve. On re-investigating the rat colon, we found that BMY 45778 (0.1 - 3 microM), BMY 42393 (3 microM) and ONO-1301 (3 microM) behaved as specific IP(1) partial agonists, but their actions required 30 - 60 min to reach steady-state and only slowly reversed on washing. This profile contrasted sharply with the rapid and readily reversible contractions elicited by a related non-prostanoid ONO-AP-324, which is an EP(3)-receptor agonist. The full versus partial agonism of the non-prostanoid prostacyclin mimetics may be explained by the markedly different IP(1) agonist sensitivities of the two rat neuronal preparations. However, the slow kinetics of the non-prostanoids on the NANC system of the colon remain unexplained, and must be taken into account when characterizing neuronal IP-receptors.  (+info)

Activation of PPARdelta alters lipid metabolism in db/db mice. (4/91)

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which heterodimerize with the retinoid X receptor and bind to peroxisome proliferator response elements in the promoters of regulated genes. Despite the wealth of information available on the function of PPARalpha and PPARgamma, relatively little is known about the most widely expressed PPAR subtype, PPARdelta. Here we show that treatment of insulin resistant db/db mice with the PPARdelta agonist L-165041, at doses that had no effect on either glucose or triglycerides, raised total plasma cholesterol concentrations. The increased cholesterol was primarily associated with high density lipoprotein (HDL) particles, as shown by fast protein liquid chromatography analysis. These data were corroborated by the chemical analysis of the lipoproteins isolated by ultracentrifugation, demonstrating that treatment with L-165041 produced an increase in circulating HDL without major changes in very low or low density lipoproteins. White adipose tissue lipoprotein lipase activity was reduced following treatment with the PPARdelta ligand, but was increased by a PPARgamma agonist. These data suggest both that PPARdelta is involved in the regulation of cholesterol metabolism in db/db mice and that PPARdelta ligands could potentially have therapeutic value.  (+info)

Function and distribution of beta3-adrenoceptors in rat, rabbit and human urinary bladder and external urethral sphincter. (5/91)

1. Activation of beta-adrenoceptors causes relaxation of the urinary bladder and contraction of the external urethral sphincter, which consists of fast-contracting skeletal muscles. A beta2-adrenoceptor agonist, clenbuterol, recently has been developed as a therapeutic drug for the treatment of urinary incontinence, however beta2-adrenoceptor agonists have undesirable effects on cardiac and striated muscle function. 2. In this study, we compared the effects of the beta2-adrenoceptor agonist, clenbuterol and of a novel beta3-adrenoceptor agonist, GS332, on urinary bladder and external urethral sphincter function in rat, rabbit and human. We also determined the distribution of beta3-adrenoceptors in human urinary bladder and external urethral sphincter, using radioligand-binding techniques. 3. Clenbuterol induced marked relaxations in rat, rabbit and human urinary bladder smooth muscles and also induced marked contractions in rat periurethral striated muscles (external urethral sphincter), while GS332 induced marked relaxations in rat and human, but not in rabbit, urinary bladder smooth muscles and induced small contractions in rat periurethral striated muscles. 4. The radioligand binding studies showed presence of beta2- and beta3-adrenoceptors in human urinary bladder, external urethral sphincter and abdominal rectus muscles. The affinities of GS332 were the highest in urinary bladder and the lowest in the skeletal (abdominal rectus) muscles, while the affinities of clenbuterol were similar in urinary bladder, external urethral sphincter and the skeletal (abdominal rectus) muscles. 5. These results suggest that GS332 could, similarly clenbuterol, have a role in the treatment of urinary frequency and urinary incontinence.  (+info)

Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament. (6/91)

To determine the ability of strong-binding myosin cross-bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross-bridge interaction kinetics at 15 degrees C in the absence and presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1) in chemically skinned myocardium from adult rats. Relative to control conditions, application of 6 microM NEM-S1 significantly increased Ca2+-independent tension, measured at pCa 9.0, from 0.8 +/- 0.3 to 3.7 +/- 0.8 mN mm-2. Furthermore, NEM-S1 potentiated submaximal Ca2+-activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half-maximal activation (pCa50) increased from pCa 5.85 +/- 0.05 to 5.95 +/- 0.04 (change in pCa50 (dpCa50) = 0.11 +/- 0.02). The augmentation of submaximal force by NEM-S1 was accompanied by a marked reduction in the steepness of the force-pCa relationship for forces less than 0.50 Po (maximum Ca2+-activated force), i.e. the Hill coefficient (n2) decreased from 4.72 +/- 0.38 to 1.54 +/- 0.07. In the absence of NEM-S1, the rate of force redevelopment (ktr) was found to increase from 1.11 +/- 0.21 s-1 at submaximal [Ca2+] (pCa 6.0) to 9.28 +/- 0.41 s-1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM-S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 +/- 0.30 s-1 at pCa 6.6 compared to 9.23 +/- 0.27 s-1 at pCa 4. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM-S1. NEM-S1 dramatically reduced both the extent and rate of relaxation from steady-state submaximal force following flash photolysis of the caged Ca2+ chelator diazo-2. These data demonstrate that strongly bound myosin cross-bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross-bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.  (+info)

Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle. (7/91)

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.  (+info)

Effect of postnatal age and a beta(3)-adrenergic agonist (Zeneca D7114) administration on uncoupling protein-1 abundance in the lamb. (8/91)

We examined the effect of time after birth and beta(3)-adrenergic agonist (Zeneca D7114) administration on uncoupling protein-1 (UCP1) abundance and thermoregulation in the lamb. Forty twin lambs, all born normally at term, were maintained at a cold ambient temperature of between 3 and 8 degrees C. At 0.5, 1.75, 5.25, 11.25 and 23.25 h after birth eight sets of twins were fed 20 ml of formula milk +/- 10 mg kg(-1) of beta(3)-adrenergic agonist, and 45 min after feeding brown adipose tissue (BAT) was sampled. Colonic temperature was measured and BAT analysed for UCP1 abundance, GDP-binding to mitochondrial protein (i.e. thermogenic activity) and catecholamine content. Colonic temperature declined between 1.25 and 6 h from 40.2 degrees C to 39.2 degrees C and then increased to 39.8 degrees C at 12 h, but increased after feeding at all ages. UCP1 abundance increased from 1.25 h after birth, to peak at 2 h after birth in controls, compared with 6 h after birth in beta(3)-adrenergic agonist-treated lambs. The level of GDP-binding to mitochondrial protein did not change significantly with age but was increased by beta(3)-adrenergic agonist treatment. The noradrenaline (norepinephrine) content of BAT increased between 1.25 and 12 h after birth, irrespective of beta(3)-adrenergic agonist administration. The total weight of perirenal BAT plus its lipid, protein and mitochondrial protein content declined over the first 6 h of life. UCP1 development continues over the first 24 h of neonatal life, and can be manipulated by beta(3)-adrenergic agonist administration. This may represent one method of improving thermoregulation in newborn lambs. Experimental Physiology (2001) 86.1, 65-70.  (+info)