Norma L. Gómez-Viquez

Inhibition of SERCA pumps induces desynchronized RyR activation in overloaded internal Ca2+ stores in smooth muscle cells

We have previously shown that rapid inhibition of sarcoplasmic reticulum (SR) ATPase (SERCA pumps) decreases the amplitude and rate of rise (synchronization) of caffeine induced-Ca(2+) release without producing a reduction of free luminal SR Ca(2+) level in smooth muscle cells (Gómez-Viquez L, Guerrero-Serna G, García U, Guerrero-Hernández A. Biophys J 85: 370-380, 2003). Our aim was to investigate the role of luminal SR Ca(2+) content in the communication between ryanodine receptors (RyRs) and SERCA pumps. To this end, we studied the effect of SERCA pump inhibition on RyR-mediated Ca(2+) release in smooth muscle cells with overloaded SR Ca(2+) stores. Under this condition, the amplitude of RyR-mediated Ca(2+) release was not affected but the rate of rise was still decreased. In addition, the caffeine-induced Ca(2+)-dependent K(+) outward currents revealed individual events, suggesting that SERCA pump inhibition reduces the coordinated activation of RyRs. Collectively, our results indicate that SERCA pumps facilitate the activation of RyRs by a mechanism that does not involve the regulation of SR Ca(2+) content. Importantly, SERCA pumps and RyRs colocalize in smooth muscle cells, suggesting a possible local communication between these two proteins.

Chronic potentiation of cardiac L-type Ca2+ channels by pirfenidone

AIMS On the basis of its ability to inhibit fibrosis, pirfenidone has drawn the attention as an intriguing candidate for treating cardiac disease. However, its precise electrophysiological effects have yet to be elucidated. Here, we have investigated its potential to modulate ion channels. METHODS AND RESULTS Adult rat cardiac myocytes were investigated using whole-cell patch-clamp, western-blot and qRT-PCR techniques. Pirfenidone increased the density of L-type Ca(2+) current (I(CaL,) 50-100%), without significantly altering Na(+), K(+), or T-type Ca(2+) currents. The effect was dose-dependent, with an EC(50) of 2.8 µM. Its onset was slow, with a lag period larger than 1 h and time to maximum of 24-48 h. Concomitant changes were observed in the voltage-dependent activation of I(CaL) (-5 mV shift in both V(1/2) and k). In contrast, the following properties of I(CaL) remained normal: steady-state inactivation, Ca(V)1.2 levels (mRNA and protein), and intramembrane charge movement. Indeed, the conductance-to-charge ratio, or G(max)/Q(max), was increased by 80%. The effect on I(CaL) was mimicked by an inhibitor of nitric oxide (NO) synthase (NOS), and attenuated by both cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) inhibitors. Conversely, cytokines, reactive oxygen species, and Ca(2+) were all ruled out as possible intermediaries. Additional experiments suggest that pirfenidone increases action potential duration by ∼50%. CONCLUSION Pirfenidone augments I(CaL), not through higher expression of L-type channels, but through promoting their Ca(2+)-conducting activity. A possible inhibition of NOS expression is likely involved, with subsequent reduced NO production and stimulated cAMP/PKA signalling. These findings may be relevant to the cardioprotective effect of pirfenidone.

Role of alpha adrenoceptors and nitric oxide on cardiovascular responses in acute and chronic hypertension

The contribution of α-adrenoceptors and nitric oxide (NO) on the alterations of sympathetically mediated cardiovascular responses after acute (AcH) and chronic (ChH) hypertension was evaluated in pithed aortic coarcted hypertensive rats. Pressor and tachycardia response produced by electrical stimulation of preganglionic sympathetic fibers or exogenous noradrenaline (NA) were recorded in the absence and presence of prazosin (α1-antagonist), rauwolscine (α2-antagonist), or NG-nitro-l-arginine methyl ester (l-NAME; an inhibitor of NO synthase). Compared with age-matched sham-operated rats (Nt), the pressor response produced by electrical stimulation or NA was smaller in AcH rats and larger in ChH rats. Prazosin caused a decrease of pressor response elicited by electrical stimulation or NA in all groups. However, this effect was higher in ChH. Rauwolscine produced a similar increase of sympathetically mediated pressor response in Nt and AcH rats. Nevertheless, this antagonist did not affect the sympathetically mediated pressor response in ChH rats. In addition, rauwolscine did not affect the NA-induced pressor response in all groups. The pressor response elicited by l-NAME was larger in all groups compared without l-NAME and in presence of l-arginine. Moreover, l-NAME in the presence of NA increased sympathetically mediated pressor response is in all groups, compared without it or in the presence of l-arginine. Compared with Nt, basally produced NO in aortic rings was increased in AcH but decreased in ChH. Collectively, our data suggest that decreased cardiovascular reactivity in AcH is due to an increase in basally produced NO. In ChH, enhanced cardiovascular response appears to be associated with a decrease in produced NO and an increase in released NA from sympathetic nerves.

Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes

In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

Evidence that chronic administration of 17β-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-diabetic female rats

In vitro studies have indicated that 17β-oestradiol exerts beneficial effects on the cardiovascular system by activating the nitric oxide pathway. However, these effects have not been demonstrated in vivo in the systemic vasculature of rats made diabetic through streptozotocin induction. Therefore, the goal of this study was to determine the effect of 17β-oestradiol on vasopressor responses induced by sympathetic stimulation or i.v. injections of noradrenaline, methoxamine and B-HT 933 in sham-operated or ovariectomised, diabetic or non-diabetic female rats. Thus, rats were ovariectomised or sham-operated for this experiment. One week later, the animals were treated with streptozotocin (60mg/kg, i.p.) or its vehicle. Two weeks later, these rats were treated daily with 17β-oestradiol (10μg/kg, s.c.) or its vehicle for five weeks. Next, under anaesthesia, the animals were pithed and prepared for blood pressure and heart rate measurements. 17β-oestradiol failed to modify the vasopressor responses to (i) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in sham-operated non-diabetic rats; (ii) sympathetic stimulation or B-HT 933 in sham-operated diabetic rats; (iii) noradrenaline or methoxamine in ovariectomised non-diabetic rats. In contrast, 17β-oestradiol significantly decreased the vasopressor responses to (i) noradrenaline and methoxamine in sham-operated diabetic rats; (ii) sympathetic stimulation or B-HT 933 in ovariectomised non-diabetic rats; and (iii) sympathetic stimulation, noradrenaline, methoxamine or B-HT 933 in ovariectomised diabetic rats. These results suggest that chronic administration of 17β-oestradiol decreases the vasopressor responses to adrenergic system stimulation in streptozotocin-induced diabetic rats. This report describes the first in vivo study reporting this effect of 17β-oestradiol in diabetes.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.

Beneficial effects of lipidic extracts of saladette tomato pomace and Serenoa repens on prostate and bladder health in obese male Wistar rats: Effects of natural extracts on prostate and bladder health in obese rats

BACKGROUND Obesity is associated with increased risk of a number of serious medical conditions, including urological disorders. This study investigated the effect of lipidic extracts of saladette tomato pomace (STP) and Serenoa repens (SR) on the prostate and bladder in a rat obese model induced by high-carbohydrate diet. RESULTS High-sucrose-fed rats showed higher prostate weight as well as increased contractility and stromal and epithelial hyperplasia in the prostate. Treatment with STP and SR improved contractility and diminished hyperplasia and hypertrophy in the prostate. Obese animals also showed impaired bladder contractility, but neither extract reversed this deterioration. In the histological study, a disarray in the process of smooth muscle cell proliferation with non-parallel fibers was observed; interestingly, treatment with STP and SR led to improvement in this derangement. CONCLUSION These findings indicated impaired contractility and hyperplasia in the prostate and bladder of obese rats induced by high sucrose. STP and SR could enhance prostate function by reducing contractility and hyperplasia and improve smooth muscle fiber structure and decrease cell proliferation in the bladder, suggesting their possible health-beneficial effects on lower urinary tract symptoms.

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells.

The process of Ca2+ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca2+ concentration ([Ca2+]i) with a minimal reduction of the free luminal SR [Ca2+] ([Ca2+]FSR). Importantly, active SR Ca2+ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca2+ binding sites. A new mathematical model was developed that assumes an increasing SR Ca2+ buffering capacity in response to an increase of the luminal SR [Ca2+] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca2+]FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca2+ binding proteins switch to standard kinetics. The importance of KonD Ca2+ binding properties is twofold: a more efficient Ca2+ release process and that [Ca2+]FSR and Ca2+-bound to SR proteins ([Ca2+]BSR) can be regulated separately allowing for Ca2+ release to occur (provided by Ca2+-bound to luminal Ca2+ binding proteins) without an initial reduction of the [Ca2+]FSR.