Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.     

Alpha(1)- and beta-adrenoceptor-mediated increase in 86Rb(+)-uptake in isolated cardiomyocytes from adult rat heart: evidence for interaction between the two receptor systems

The aim of the present investigation was to study the effect of alpha(1)- and beta-adrenoceptor stimulation, alone and in combination, on potassium uptake in isolated ventricular cardiomyocytes from adult rat heart, using the potassium analogue 86Rb+. The reliability of 86Rb+ as a potassium analogue was also investigated. Alpha(1)-, beta-, And combined adrenoceptor stimulation was achieved by using noradrenaline in the presence and absence of appropriate adrenoceptor antagonists. The uptake of 86Rb+ was found to increase linearly with time up to 20 min., both during basal and receptor-stimulated conditions. The basal uptake rate was about 0.18 ml/g protein x min. At 15 min. both alpha(1)-, beta- and combined adrenoceptor stimulation dose-dependently increased the 86Rb(+)-uptake with a -logEC50 of 7.05, 6.68 and 6.73, respectively. The maximal increase in these series achieved by 5 x 10(-5) mol/l noradrenaline was 29%, 24% and 41% above basal level, respectively. Comparison of the maximal effects in the same cell preparations, with the observed value for combined adrenoceptor stimulation in each experiment as 100%, gave a relative maximal increase in 86Rb(+)-uptake after separate alpha(1)-adrenoceptor stimulation of 67 +/- 8%, and of 68 +/- 6% after separate beta-adrenoceptor stimulation. The theoretically calculated value for combined adrenoceptor stimulation, if additivity, was 135 +/- 11%, which was significantly higher than the observed value (100%) (P = 0.026). The effect of noradrenaline was not limited by the maximal 86Rb(+)-uptake capacity, as 10(-5) mol/l forskolin increased the 86Rb(+)-uptake more than noradrenaline. Examining the reliability of 86Rb+ as potassium-analogue by combining 42K+ and 86Rb+ in the same experiments, showed that combined adrenoceptor stimulation dose-dependently increased both the 42K(+)- and 86Rb(+)-uptake with the same potency and to the same extent. Thus 86Rb+ is a reliable potassium-analogue for these effects. In conclusion both alpha(1)- and beta-adrenoceptor stimulation dose-dependently increased the cellular 86Rb(+)-uptake to the same extent and with the same potency. The observed maximal 86Rb(+)-uptake after combined adrenoceptor stimulation was significantly higher than the maximal effect after either form of separate receptor stimulation, but significantly lower than expected if the effects were purely additive. The results thus show inhibitory interaction between the two receptor systems.     

Inhibition by protein kinase C of the 86Rb+ efflux and vasorelaxation induced by P1075, a K(ATP) channel opener, in rat isolated aorta

The expression of GABA in the human fetal (12-25 weeks of gestation), postnatal (five-month-old), and adult (35-year-old) retinas was investigated by immunohistochemistry. GABA expression was seen as early as 12 weeks in the undifferentiated cells of the inner neuroblast zone; a few optic nerve fiber layer axons were clearly labeled, suggesting that some of the stained cell bodies were prospective ganglion cells, others could be displaced amacrine cells. From 16-17 to 24-25 weeks, intense labeling was found in the amacrine, displaced amacrine, and some ganglion cells. During this time period, horizontal cells (identified by calbindin immunohistochemistry), undergoing migration (periphery) and differentiation (center), expressed GABA prominently. In the postnatal retina, some horizontal cells were moderately labeled, but very weakly in a few cells, in the adult. The Müller cells developed immunoreactivity first weakly at 12 weeks and then moderately from 16-17 weeks onward. The staining was also evident in the postnatal and adult retinas, showing labeled processes of these glial cells. Virtually no axons in the adult optic nerve and nerve fiber layer were stained; the staining was restricted to a few, large ganglion cells and displaced amacrine cells: Some amacrines were also labeled. The possibility that GABA might play a role in horizontal cell differentiation and maturation is highlighted. Other evidences suggest that GABA might play a role in metabolism during retinal development.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Sustained increase in rat myocardial alpha 1A-adrenoceptors induced by 6-hydroxydopamine treatment involves a decelerated receptor turnover

The biochemical mechanisms involved in the alpha 1-adrenoceptor up-regulation and possible changes in subtypes of adrenoceptors in the rat heart after chemical denervation were investigated. The effects of acute 6-hydroxydopamine treatment (two increasing doses 24 h apart) on the pseudo-steady state densities and turnover rates of alpha 1-adrenoceptors were studied in ventricular myocardium of the rat. We have assessed the repopulation kinetics of [3H]prazosin binding sites after irreversible inactivation of alpha 1-adrenoceptors induced by a single dose of phenoxybenzamine (1 mg/kg i.p.) in rats acutely treated either with 6-hydroxy-dopamine or with vehicle (control animals). Seven days after the last administration of 6-hydroxydopamine an enhanced density of [3H]prazosin binding sites (Bmax 58.7 +/- 3.6 fmol/mg protein vehicle-treated rats versus 82.6 +/- 5.3 fmol/mg protein 6-hydroxydopamine-treated rats) was observed. This was not accompanied by changes in the dissociation constant value. Furthermore, the proportion of high affinity sites for WB-4101 was altered (21 +/- 2% versus 72 +/- 3% for animals treated with vehicle and 6-hydroxydopamine, respectively). In rat myocardium, alpha 1-adrenoceptor turnover, evaluated during the 6-hydroxydopamine-induced up-regulation (7-19 days after the completion of treatment with 6-hydroxydopamine) revealed an increase in the half-life of the alpha 1-adrenoceptor (t1/2 of 67.2 h versus 38.7 h in control animals). The present study confirms an increase in alpha 1-adrenoceptors in rat myocardium after chemical denervation and reveals that the effect is almost completely confined to the alpha 1A-adrenoceptor subtype. Furthermore, the up-regulation of alpha 1A-adrenoceptors is the result of a decrease in the cellular processes that control the rate of receptor degradation.     

Natriuretic peptides increase a K+ conductance in rat mesangial cells

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3',5'-cyclicmonophosphate) peptides on membrane voltages (Vm) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/l. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on Vm these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodalatin) hyperpolarized Vm significantly by 1.6 +/- 0.4 mV (BNP, n=8), 3.7 +/- 0.3 mV (CDD/ANP-99-126, n=25) and 2.8 +/- 0.4 mV (urodilatin, n=9), respectively. The half-maximally effective concentration (EC50) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/l 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/l Ba2+. The K+ channel blocker 293 B (100 micromol/l) depolarized basal Vm by 4.3 +/- 0.4 mV (n=8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/l) (n=8). The K+ channel opener cromakalim (10 micromol/l) neither influenced basal Vm nor altered the hyperpolarization induced by 160 nmol/l CDD/ANP-99-126 (n=8). Adenosine (100 micromol/l) hyperpolarized Vm by 13.4 +/- 1.3 mV (n=16). At 100 micromol/l, 293 B did not inhibit the adenosine-induced hyperpolarization (n=6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 +/- 0.6 mV (n=10). CDD/ANP-99-126 (160 nmol/l) failed to modulate the value to which Vm depolarized in the presence of 1 nmol/l angiotensin II (n=10), but accelerated the repolarization to basal Vm by 49 +/- 20% (n=8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K+ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba2+, but not by 293 B and cannot be activated by cromakalim. This increase in the K+ conductance seems to be additive to that inducable by adenosine, indicating that different K+ channels are activated by these hormones.     

Chronic treatment with prazosin causes a subtype-specific increase in the alpha 1-adrenoceptor density of the stressed rat cerebral cortex

The effects of chronic treatment with prazosin and of immobilization stress on the alpha 1-adrenoceptor subtypes in rat cerebral cortex have been examined. Prazosin-treated rats were allowed free access to tap water containing two different concentrations of prazosin (16 or 156 mg L-1) for 5 weeks. The mean plasma concentrations of prazosin were 5 ng mL-1 in groups treated with a low dose and 8 or 14 ng mL-1 in those treated with a high dose. Immobilization stress (2 h day-1, 2 weeks) or chronic treatment with a low dose of prazosin caused no significant change in the affinity for [3H]prazosin or in the maximum number of alpha 1-adrenoceptor sites (Bmax). However, treatment with prazosin (low dose) combined with stress increased the density of alpha 1-adrenoceptors with low affinity for prazosin. Treatment with a high dose of prazosin increased the density of alpha 1L-adrenoceptors, irrespective of stress loading. The densities of alpha 1A- and alpha 1B-adrenoceptors with high affinity for prazosin were increased only after treatment with a high dose of prazosin in combination with stress. These results indicate that three distinct alpha 1-adrenoceptor subtypes, alpha 1A, alpha 1B and alpha 1L, might be affected differently by treatment with prazosin and by stress.     

Expressed alpha 1-adrenoceptors in adult rat brown adipocytes are primarily of alpha 1A subtype

The load distribution between two internal spinal fixation devices depends, besides other factors, on their stiffness. The stiffness ranges were determined experimentally for the clamps of the AO internal fixator with lateral nut and with posterior nut as well as for the clamps of the SOCON fixator. The stiffness of eight devices each differed by a factor of 3.1 for the clamp with lateral nut, by a factor of 1.5 for the clamp with posterior nut, and by a factor of 1.4 for the clamp of the SOCON fixator. For the AO clamp with lateral nut, the influence of the nut-tightening torque on the stiffness was determined. Using instrumented internal spinal fixation devices mounted to plastic vertebrae and simulating a corpectomy, the load distribution between the implants was measured for different tightening torques. It could be shown that, for the AO internal fixator whose clamps have a lateral nut, a nut-tightening torque > 5 Nm has only a negligible influence on load-sharing between the implants. Tooth damage occurs when the teeth of the clamp body and clamping jaw of the clamp with lateral nut do not gear together exactly, which leads to changes in the clamping stiffness and load-sharing between the two implants.     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degreesC. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.     

Selective enrichment with alpha 1A- and alpha 1B-adrenoceptor subtypes in rat brain cortical membranes

In this controlled, prospective and partially blind study two groups of patients with temporal lobe epilepsy were evaluated, respectively with good and bad prognosis. Measurements of epileptogenesis were based on frequency of seizures, and on epiletogenic electroencephalographic abnormalities obtained from scalp electrodes over the temporal lobes. The results were analysed by non-parametric analysis of variance, comparison of groups and analysis of correlation. The results indicated the temporal lobe groups were similar to at least one of the control groups in age, sex, educational and social level, therapeutic regime, age at onset and length of history of epilepsy. The quantitative measurements showed a global difference between the group of temporal lobe with bad and good prognosis, reaching statistical significance in clinical epileptogenesis, and a trend towards greater epileptogenesis on the electroencephalogram, in the same group of patients. The results indicate the experimental usefulness of some of the original measurements used in the study, but also their problems. A review of the literature is carried out.     

The left ventricular contractility of the rat heart is modulated by changes in flow and alpha 1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on alpha 1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo alpha 1-adrenergic stimulation increases left ventricular developed pressure (P < 0.05) together with arterial blood pressure (P < 0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P < 0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P < 0.01). The largest ISP increase was obtained with PE + NP treatment (P < 0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial alpha 1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.     

Alpha1-adrenergic stimulation of sarcolemmal Na+-H+ exchanger activity in rat ventricular myocytes: evidence for selective mediation by the alpha1A-adrenoceptor subtype

Alpha1-adrenoceptor (alpha1-AR) stimulation increases sarcolemmal Na+-H+ exchanger (NHE) activity. The present study was designed to determine the role(s) of alpha1-AR subtype(s) in mediating this response. As an index of NHE activity, acid efflux rates (JHs) were determined in single rat ventricular myocytes loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after 2 consecutive intracellular acid pulses in bicarbonate-free medium. JH at pHi 6.90 did not change significantly during the second pulse relative to the first in control cells but increased in a dose-dependent manner when the second pulse occurred in the presence of phenylephrine (nonselective alpha1-AR agonist) or A61603 (alpha1A-AR-selective agonist), with EC50 values of 1.24 micromol/L and 3.6 nmol/L, respectively (both agonists given together with 1 micromol/L atenolol). Stimulation of NHE activity by 10 micromol/L phenylephrine was inhibited in a dose-dependent manner by the competitive antagonists prazosin, WB4101, and 5-methylurapidil, with IC50 values of 12, 32, and 149 nmol/L, respectively. Analyses of the relative EC50 and IC50 values obtained (and Ki values estimated from the antagonist IC50s) in relation to the relative potencies of these agents at native rat alpha1-AR subtypes and their relative affinities for recombinant rat alpha1-ARs suggest that alpha1-adrenergic stimulation of sarcolemmal NHE activity is likely to be mediated selectively by the alpha1A-AR.     

Ca2+-dependence of vasoconstriction mediated by alpha1A-adrenoceptors in perfused rat hindlimb: a pharmacological approach

We investigated the source of Ca2+ for the vasoconstriction mediated by alpha1a-adrenoceptors in perfused rat hindlimb in functional studies. The noradrenaline (NA)-induced maximum response was decreased by 92% following perfusion with Ca2+-free medium. Depletion of intracellular Ca2+-stores with repeatedly application of caffeine and NA in Ca2+-free medium resulted in complete abolishment of NA-response. Nifedipine concentration-dependently inhibited NA-contraction with a maximum inhibition of 65%. The residual nifedipine-insensitive response was further inhibited by Cd2+. Following depletion of Ca2+ stores with cyclopiazonic acid in Ca2+ free medium for 30 min, the NA-response obtained by re-admission of Ca2+ was decreased by 80%. However, re-introduction of Ca2+ to NA-treated tissues in Ca2+-free medium without prior treatment with cyclopiazonic acid normalizes the NA-response. These results suggest that the NA-contraction in this preparation is mediated largely via an influx of extracellular Ca2+, of which the majority utilizes L-type calcium channels. Only a small portion of the contractile response to NA is derived from intracellular stores, which probably also play a modulatory role on Ca2+ influx.     

Modulation of oxidative phosphorylation by Mg2+ in rat heart mitochondria

The effect of varying the Mg2+ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K0.5) of ATP synthesis by Mg2+ was obtained with 0.13 +/- 0.02 mM (n = 7) with succinate (+rotenone) and 0.48 +/- 0.13 mM (n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Pi concentration promoted a decrease in the K0.5 values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.     

Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 microM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 microM PCP in vitro. A significant reduction of SR Ca2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca2+ activation. These results indicate that PCP alters the myocardial Ca2+ homeostasis by inhibiting the Ca2+ ATPase in cardiac SR in rats. Inhibition of SR Ca2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.     

Immunohistochemical localisation of alpha1B-adrenergic receptors in the rat iris

This article reports results from a meta-analysis on adult age differences in the negative priming effect (21 studies on identity negative priming and 8 on location negative priming). Both younger and older adults were found to be susceptible to the negative priming effect in identity and location tasks. Effect sizes were homogeneous for both tasks, indicating that the data are adequately described without reference to moderator variables. State trace analysis on identity tasks, in which mean latencies in negative priming conditions were regressed onto mean latencies in baseline conditions, showed (a) that in both age groups the negative priming effect is proportional rather than additive and (b) that the negative priming effect is smaller in older adults as compared with younger adults.     

The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.