Virendra K. Sharma

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca2+ uniporter (CaUP) to highconcentrations of Ca2+ following Ca2+ release from the SR and thus an influx of Ca2+into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca2+ concentration ([Ca2+]c)and mitochondrial Ca2+ concentration ([Ca2+]m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca2+]c and [Ca2+]m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca2+]m but not[Ca2+]c. In the presence of 1 mM BAPTA, a Ca2+ chelator,the caffeine-induced increase in [Ca2+]m was reduced substantially less than [Ca2+]c. Moreover,inhibition of SR Ca2+ pump with two different concentrations of thapsigargin caused anincrease in [Ca2+]m, which was related to the rate of [Ca2+]c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca2+ is released,or Ca2+ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca2+ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca2+ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.

Losartan Metabolite EXP3179 Activates Akt and Endothelial Nitric Oxide Synthase via Vascular Endothelial Growth Factor Receptor-2 in Endothelial Cells Angiotensin II Type 1 Receptor–Independent Effects of EXP3179

Background—Recent studies suggest that angiotensin type 1 receptor (AT1R) blockers have vascular protective effects beyond blood pressure lowering. Because of the importance of endothelial nitric oxide synthase (eNOS) in vascular and platelet function, we hypothesized that losartan and its metabolites would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3K). Methods and Results—Losartan is metabolized into EXP3174 (AT1R-blocking metabolite) and EXP3179 (no AT1R-blocking properties). Treatment of endothelial cells (ECs) with losartan and both metabolites stimulated phosphorylation of Akt and eNOS in the absence of angiotensin II. However, the magnitude for EXP3179 was much greater than EXP3174, and the EC50 was significantly lower (−logEC50, 8.2±0.1 versus 5.4±0.2 mol/L), suggesting an AT1R-independent effect. Inhibiting PI3K or vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine phosphorylation abrogated EXP3179-induced eNOS phosphorylation. In endothelium of intact rat aorta, EXP3179 also stimulated Akt and eNOS phosphorylation. VEGFR2 activation was shown to be calcium and Src family kinase dependent by use of specific drug inhibitors and dominant negative kinase transfection. EXP3179 significantly inhibited tumor necrosis factor &agr;–induced apoptosis by ≈60% (from 30.1±5.8% to 12.2±2.0% TUNEL-positive cells), which was abolished by pretreatment with the PI3K inhibitor LY294002. Cleaved caspase-3 was suppressed by 48% with EXP3179. Conclusions—The losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses tumor necrosis factor &agr;–induced EC apoptosis by activating the VEGFR2/PI3K/Akt pathway.

Inhibiting p90 Ribosomal S6 Kinase Prevents Na+-H+ Exchanger–Mediated Cardiac Ischemia-Reperfusion Injury

Background— Pharmacological and genetic studies indicate that the Na+-H+ exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. We found that p90 ribosomal S6 kinase (RSK) phosphorylated serine 703 of NHE1, stimulating 14–3–3 binding and NHE1 activity. Therefore, we hypothesized that inhibiting RSK in cardiomyocytes would prevent NHE1 activation and decrease I/R-mediated injury. Methods and Results— To examine the role of RSK in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative RSK (DN-RSK-TG). DN-RSK-TG hearts demonstrated normal basal cardiac function and morphology. However, myocardial infarction (left coronary artery occlusion for 45 minutes) in DN-RSK-TG hearts was significantly reduced at 24 hours of reperfusion from 46.9±5.6% area at risk in nontransgenic littermate controls to 26.0±4.2% in DN-RSK-TG (P<0.01). Cardiomyocyte apoptosis was significantly reduced after I/R in DN-RSK (0.9±0.2%) compared with nontransgenic littermate controls (6.2±2.6%). Importantly, activation of RSK and interaction of 14–3–3 with NHE1, necessary for agonist-stimulated NHE1 activity, were increased by I/R and inhibited by 70% in DN-RSK-TG (P<0.01). Next, we transduced rat neonatal cardiomyocytes with adenovirus-expressing DN-RSK (Ad.DN-RSK) and measured NHE1 activity. The baseline rate of pH recovery in acid-loaded cells was equal in cells expressing LacZ or DN-RSK. However, NHE1 activation by 100 &mgr;mol/L H2O2 was significantly inhibited in cells expressing DN-RSK (0.16±0.02 pH units/min) compared with Ad.LacZ (0.49±0.13 pH units/min). Apoptosis induced by 12 hours of anoxia followed by 24 hours’ reoxygenation was significantly reduced in cells expressing Ad.DN-RSK (18.6±2.0%) compared with Ad.LacZ (29.3±5.4%). Conclusions— In summary, RSK is a novel regulator of cardiac NHE1 activity by phosphorylating NHE1 serine 703 and a new pathological mediator of I/R injury in the heart.

Molecular and Functional Identification of m1 Muscarinic Acetylcholine Receptors in Rat Ventricular Myocytes

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol ( > 10 mumol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mumol/L) increased the basal Ca2+ level from 96 +/- 7 to 118 +/- 8 nmol/L and the peak Ca2+ transient level from 519 +/- 32 to 640 +/- 36 nmol/L (mean +/- SEM P < .05 for both, n = 8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m1 mAChR-selective antagonist. In contrast, the m2 mAChR-selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m1 mAChRs are expressed on adult rat ventricular myocytes in addition to m2 mAChRs. The results further suggest that m1 mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.

Cocaine andd-amphetamine induce changes in central ß-adrenoceptor sensitivity: Effects of acute and chronic drug treatment

The effects of acute and chronic treatment with psychomotor stimulants on specific binding of [3H]dihydroalprenolol to beta-adrenoceptors in rat brain were examined. At a dose of 10 mg/kg both acute and chronic treatment with cocaine and chronic treatment with D-amphetamine (10 mg/kg) caused increased binding of [3H]dihydroalprenolol. The molecular mechanism for this enhanced binding appears to be augmentation of the density of beta-adrenoceptors in rat brain. At a lower dose (5 mg/kg), however, chronic administration of D-amphetamine caused a decrease in the density of beta-adrenoceptors in rat brain. Chronic treatment with either D-amphetamine (10 mg/kg) or cocaine induced a marked increase in the magnitude of cyclic AMP accumulation in rat brain slices elicited by norepinephrine. Acute as well as chronic administration of D-amphetamine in vivo inhibited the temperature-dependent uptake of [3H]norepinephrine in rat brain synaptosomal homogenates, but no such inhibition was observed after chronic or acute treatment with cocaine. The results suggest that psychomotor stimulants induce beta-adrenoceptor supersensitivity which may be involved in the phenomenon of reverse tolerance and possibly psychosis in humans. The development of beta-adrenoceptor supersensitivity does not appear to be mediated through alterations in norepinephrine transport at the presynaptic sites.

Amphetamine induces beta-adrenergic receptor supersensitivity.

THE interactions of amphetamines and catecholamines in the peripheral sympathetic nervous system and in the brain have been widely studied. Amphetamines have been reported to mimic catecholamines at their receptor sites1–3, inhibit monoamine oxidase4, impair reuptake mechanism for the catecholamines5 and to directly release catecholamines into the synaptic cleft6. Although the consensus of the available literature indicates that d-amphetamine indirectly stimulates catecholamine postsynaptic receptors by increasing the release and blocking the reuptake of catecholamines7,8, the question whether dopamine, noradrenaline or both, are of importance for the central actions of d-amphetamine is controversial7,8. Recently, α-adrenergic9, β-adrenergic10–13 and dopaminergic14,15 receptors in brain tissue have been successfully identified by measuring the binding of radiolabelled ligands to specific receptor sites. Availability of such methods has permitted the examination of the effects of acute and chronic administration of psychotropic drugs on the postsynaptic catecholaminergic receptors in brain tissue13,16. We report here the effects of acute and chronic administration of d-amphetamine on the postsynaptic β-adrenergic receptors in rat brain.

Mitochondria regulate inactivation of L‐type Ca2+ channels in rat heart

1 L‐type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage‐dependent and a Ca2+‐dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague‐Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m‐chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2 RR and CCCP also caused a significant delay in the recovery from inactivation of L‐type Ca2+ currents (ICa). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L‐type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+‐dependent inactivation of L‐type Ca2+ channels. 3 We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L‐type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L‐type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro‐domain near L‐type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.

β-adrenergic receptors in rat skeletal muscle effects of thyroidectomy

Thyroid hormones may participate in the regulation of beta-adrenergic receptors in skeletal muscle sarcolemmal membrane. Since skeletal muscles are not innervated by sympathetic nerve endings, the biochemical mechanism involved in the control of beta-adrenergic receptors by thyroid hormones appears to be mediated by thyroid-induced regulation of serum levels of catecholamines.

Signaling Mechanisms Underlying Muscarinic Receptor-mediated Increase in Contraction Rate in Cultured Heart Cells

We have investigated the mechanisms by which stimulation of cardiac muscarinic receptors result in paradoxical stimulatory effects on cardiac function, using cultured neonatal rat ventricular myocytes as a model system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nm) produced an atropine-sensitive decrease in spontaneous contraction rate, while, in cells pretreated with pertussis toxin, higher concentrations of CCh (EC50 = 26 μm) elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor (mAChR) agonist, mimicked the negative but not the positive chronotropic response to CCh. Reverse transcription followed by polymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m1, m2, and, possibly, m4 mAChRs. The presence of m1 and m2 mAChR protein on the surface membranes of the cultured ventricular myocytes was confirmed by immunofluorescence. The CCh-induced positive chronotropic response was significantly inhibited by fluorescein-tagged antisense oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was also inhibited by antisense oligonucleotides against Gqα protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis with 500 μm neomycin or 5 μm U73122 completely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activation on the rate of contractions in cultured ventricular myocytes being mediated through the m1 mAChR coupled through Gq to phospholipase C-induced phosphoinositide hydrolysis.

Effects of noradrenaline depletion on adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum

Abstract The effects of noradrenaline depletion on α- and β-adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum of Wistar rats were studied. Noradrenaline depletion was obtained either by chemical (6-hydroxydopamine) lesions of the locus cerulets or by chronic reserpine treatment. Two weeks after locus ceruleus lesions and 10 days after chronic reserpine treatment, the rats were killed and the cerebral cortex, hippocampus, and cerebellum dissected. A small portion of each tissue was assayed for noradrenaline, to assess the success of locus ceruleus lesions or reserpine treatment, and the remainder was used to measure the specific binding of tritiated α- and β-adrenergic and muscarinic cholinergic receptor ligands to particulate fractions of these tissues. The effect of noradrenaline depletion on isoproterenol-induced cyclic AMP generation in cerebellar slices was also studied. Noradrenaline depletion whether by locus ceruleus lesion or chronic reserpine treatment induced reproducible and significant increases in the binding to β-adrenergic receptors in the cerebral cortex and hippocampus. Scatchard analyses revealed that this increased binding was due to an increased density of β-adrenergic receptor binding sites. In the cerebellum, however, noradrenaline depletion did not result in an increase in β-adrenergic receptor binding nor in isoproterenol-induced cyclic AMP generation. Noradrenaline depletion did not cause significant changes in the binding characteristics of α-adrenergic or muscarinic cholinergic receptors in any of the three regions of the brain that were studied.