Sergio Bova

Congenital Deafness and Sinoatrial Node Dysfunction in Mice Lacking Class D L-Type Ca2+ Channels

Voltage-gated L-type Ca2+ channels (LTCCs) containing a pore-forming alpha1D subunit (D-LTCCs) are expressed in neurons and neuroendocrine cells. Their relative contribution to total L-type Ca2+ currents and their physiological role and significance as a drug target remain unknown. Therefore, we generated D-LTCC deficient mice (alpha1D-/-) that were viable with no major disturbances of glucose metabolism. alpha1D-/-mice were deaf due to the complete absence of L-type currents in cochlear inner hair cells and degeneration of outer and inner hair cells. In wild-type controls, D-LTCC-mediated currents showed low activation thresholds and slow inactivation kinetics. Electrocardiogram recordings revealed sinoatrial node dysfunction (bradycardia and arrhythmia) in alpha1D-/- mice. We conclude that alpha1D can form LTCCs with negative activation thresholds essential for normal auditory function and control of cardiac pacemaker activity.

Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels.

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

Effects of an Endogenous Ouabainlike Compound on Heart and Aorta

An endogenous ouabainlike compound (OLC) has been purified from human plasma, and mass spectrometry has shown it to be indistinguishable from plant-derived ouabain. This human OLC was tested for its effects on evoked tension in guinea pig left atria and aortic rings. The tissues were incubated at 37°C in bicarbonate-buffered physiological salt solution gassed with 95% O]-5% CO2. In atria stimulated electrically at 1 Hz, 85 and 170 nM human OLC increased peak active force to 177±15% and 313±32% of control, respectively (n=3), with little effect on the duration of contraction. On washout of the OLC, peak systolic force returned to the control level with a half-time of 4.3 ±0.5 minutes. Similar results were obtained with 160 nM plant-derived ouabain: peak systolic force increased to 310±31% of control (n=4) and returned to the control level with a half-time of 3.8 ±0.2 minutes during washout In aortic rings, neither 170 nM human OLC nor 160 nM plant ouabain (30-minute treatments) affected resting (unstimulated) tension, but they increased the contractions evoked by histamine (0.2-1.0 /μM) to 156±13% (n=4) and 143±6% (n=4) of control responses, respectively. The mean half-time for washout of the OLC and plant ouabain-induced augmentation of histamine-evoked tension exceeded 35 minutes. These data show that human OLC has cardiotonic and vasotonic actions qualitatively and quantitatively similar to those observed with plant ouabain. Together with data on the mass spectrometry, chromatographic characteristics and interaction with Na+,K+-ATPase, and full cross-reactivity with polyclonal anti-ouabain antibodies, these physiological findings support the view that human OLC is ouabain.

Positive cardiac inotropic effect of albumin infusion in rodents with cirrhosis and ascites: molecular mechanisms

The aim of this study was to evaluate the effect and molecular mechanism of albumin infusion on cardiac contractility in experimental cirrhosis with ascites. Cardiac contractility was recorded ex vivo in rats with cirrhosis and ascites and in control rats after the injection in the caudal vein of albumin, saline, or hydroxyethyl starch (HES). Gene and protein expression of β‐receptors and pathways involved in their intracellular signaling such as Gαi2 protein (Gαi2), adenylate cyclase 3 (Adcy3), protein expression of tumor necrosis factor alpha (TNF‐α) and inducible nitric oxide synthase (iNOS), were evaluated in cardiac tissue in both groups. Phosphorylation and membrane‐translocation of the cytosolic components of nicotinamide adenine dinucleotide phosphate (NAD(P)H)‐oxidase and translocation of nuclear factor kappa B (NF‐κB) were also evaluated. After saline intravenous injection, cardiac contractility was significantly reduced in rats with cirrhosis as compared to control rats (P < 0.01). This was associated with: (1) increased expression of protein Gαi2 (P < 0.05), TNF‐α (P < 0.05), iNOS (P < 0.05); (2) increased NAD(P)H‐oxidase activity (P < 0.05); (3) increased nuclear translocation of NF‐κB (P < 0.05); and (4) lower expression of Adcy 3 (P < 0.05) in cardiac tissue of rats with cirrhosis. After albumin injection cardiac contractility (P < 0.01), protein expression of TNF‐α, iNOS, Gαi2, and Adcy3, NAD(P)H‐oxidase activity and nuclear translocation of NF‐κB in cardiac tissue of rats with cirrhosis were reversed to control levels (P < 0.05). HES injection did not modify cardiac contractility and nuclear translocation of NF‐κB in cardiac tissue of rats with cirrhosis. Conclusion: Albumin exerts a positive cardiac inotropic effect in rats with cirrhosis and ascites counteracting the negative effects of oxidative stress‐ and TNF‐α‐induced activation of NF‐κB‐iNOS pathway and oxidative stress‐induced alteration of β‐receptor signaling. (HEPATOLOGY 2013)

Na/Ca exchange and tension development in vascular smooth muscle: effect of amiloride

1 The potassium‐sparing diuretic, amiloride, has been shown to inhibit the Na/Ca exchange system in various preparations. The effects of this drug have been investigated on the contractions of guinea‐pig aortic strips elicited by reduction of external K, by addition of ouabain and by removal of external Na. 2 Amiloride (5 × 10−6 m‐5 × 10−4 m) inhibited the mechanical responses when it was added before giving the stimulus for contractions, but was not effective in relaxing the contracted strips. The drug shifted to the right the dose‐response curve for Ca in low K solution. 3 The calcium antagonist diltiazem had no effect on the ouabain‐, low K‐ and Na‐free‐induced contractions. 4 Amiloride decreased the rate of relaxation of aortic strips induced by removal of the low K solution. 5 The pattern of amiloride action on ouabain‐, low K‐ and Na‐free‐induced contractions suggests that the drug interferes with Ca influx. The effect of amiloride on the relaxation rate of low K‐ contracted aortic strips is consistent with an interference with Ca efflux. 6 It is suggested that amiloride prevents Ca fluxes through the Na/Ca exchange system of guinea‐pig aortic strips.

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway.

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.

Effects of pinacidil on guinea-pig isolated perfused heart with particular reference to the proarrhythmic effect.

1 The effects of pinacidil (10, 30, 50 μm) on contractility (+ dP/dtmax), coronary perfusion pressure (cP), and ECG intervals (PR, QRS, QT) have been studied on constant‐flow perfused guinea‐pig hearts, driven at four frequencies (2.5, 3, 3.5, 4 Hz). 2 Pinacidil decreased +dP/dtmax, cP and the QT interval in a dose‐dependent manner, whereas the PR interval was increased. QRS duration was not modified. All these effects were independent of driving frequency. Pinacidil decreased the interval from Q‐wave to T‐wave peak (QTpeak) to a greater extent than the QT interval, thus decreasing the QTpeak/QT ratio. This effect, unlike that on QT interval, was more evident at the highest frequency of stimulation. 3 In 4 out of 20 hearts treated with pinacidil sustained ventricular fibrillation (VF) occurred following a short run of premature ventricular beats (R on T phenomenon). 4 In separate experiments, an attempt to induce VF electrically was made at drug concentrations ranging from 10 μm to 100 μm (8 experiments for each concentration). In control conditions and at the lowest concentration of pinacidil tested (10 μm) VF could never be induced; in the presence of 30 μm pinacidil VF was induced in 5 out of 8 experiments. Drug concentrations higher that 50 μm permitted the induction of VF in every case. 5 Although the concentrations of pinacidil producing ventricular fibrillation are 30–40 times higher than those found in patients under long term treatment with this agent, it is suggested that caution should be used in prescribing this drug, at least in patients suffering from myocardial ischaemia.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1 The effects of norbormide on the contractility of endothelium‐deprived rat, guinea‐pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2 In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5–50 μm) induced a concentration‐dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10–800 μm) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3 In resting rat and guinea‐pig aortae, guinea‐pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10–100 μm) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2+‐free medium. Norbormide (up to 100 μm) was ineffective in phenylephrine‐contracted guinea‐pig and rat aorta. 4 In A7r5 cells, a cell line from rat aorta, norbormide prevented high K+‐but not 5‐hydro‐xytryptamine‐induced intracellular calcium transients. 5 These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.

NAD+-dependent SIRT1 deactivation has a key role on ischemia–reperfusion-induced apoptosis

Ischemia-reperfusion (IR) leads to severe organ injury and dysfunction. Sirtuins (SIRTs) are a family of histone deacetylases (HDACs) that require nicotinamide adenine dinucleotide (NAD(+)) for the deacetylation reaction. SIRTs play a major role in counteracting cellular stress and apoptosis. This study aimed to investigate the mechanisms of heart protection against apoptosis by SIRTs and the molecular pathways involved in SIRTs regulation and function in a rat model of IR injury. Hearts of male Wistar-Kyoto rats were subjected to 30-min ischemia followed by reperfusion up to 6h. IR increased cardiomyocyte apoptosis; the cleavage of caspase 3, induced a transient upregulation of SIRT1 and downregulation of SIRT6 expression, but decreased SIRT1 activity and reduced NAD(+) content. IR also increased forkhead box protein O1 (FoxO1) expression and FoxO1 binding to SIRT1 promoter region. Resveratrol restored SIRT1 activity and NAD(+) level by an AMPK-dependent mechanism, reduced cardiomyocyte apoptosis, and attenuated caspase 3 cleavage via heat shock factor-1 deacetylation and heat shock protein (HSP) expression upregulation. Our data show new potential molecular mechanisms of up and downstream regulation of SIRT1 in IR. The interplay among FoxO1, SIRT1, NAD(+), AMPK, HSP, and SIRT6 depicts a complex molecular network that protects the heart from apoptosis during IR and may be susceptible to therapeutic interventions.

Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ETA and ETB) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ETA, and ETB complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ETB agonist IRL-1620; this effect was not inhibited by either the ETA antagonist BQ-123 or the ETB antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ETB, but not ETA, activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active...