Antonio Castellano

Mechanisms of Low-Glucose Sensitivity in Carotid Body Glomus Cells

OBJECTIVE—Glucose sensing is essential for the adaptive counterregulatory responses to hypoglycemia. We investigated the mechanisms underlying carotid body (CB) glomus cells activation by low glucose. RESEARCH DESIGN/METHODS AND RESULTS—Removal of extracellular glucose elicited a cell secretory response, abolished by blockade of plasma membrane Ca2+ channels, and a reversible increase in cytosolic Ca2+ concentration. These data indicated that glucopenia induces transmembrane Ca2+ influx and transmitter secretion. In patch-clamped glomus cells, exposure to low glucose resulted in inhibition of macroscopic outward K+ currents and in the generation of a depolarizing receptor potential (DRP). The DRP was abolished upon removal of extracellular Na+. The membrane-permeable 1-oleoyl-2-acetyl-sn-glycerol induced inward currents of similar characteristics as the current triggered by glucose deficiency. The functional and pharmacological analyses suggest that low glucose activates background cationic Na+-permeant channels, possibly of the transient receptor potential C subtype. Rotenone, a drug that occludes glomus cell sensitivity to hypoxia, did not abolish responsiveness to low glucose. The association of Glut2 and glucokinase, characteristic of some high glucose–sensing cells, did not seem to be needed for low glucose detection. CONCLUSIONS—Altogether, these data support the view that the CB is a multimodal chemoreceptor with a physiological role in glucose homeostasis.

Pore mutations in Shaker K+ channels distinguish between the sites of tetraethylammonium blockade and C-type inactivation.

1. We have studied the effect of external K+ and tetraethylammonium (TEA) on several mutants of Shaker B K+ channels with amino acid substitutions in the pore which alter TEA affinity and the rate of C‐type inactivation. In all channels studied high external K+ makes C‐type inactivation slower. 2. In the wild‐type channel, TEA blockade is voltage dependent and produces slowing of the inactivation time course. However, in the double mutant channel (T449Y, D447E) TEA blockade, although of higher affinity, is voltage independent and does not affect the rate of C‐type inactivation. 3. Mutants with a charged amino acid at position 449 (T449K and T449E) are resistant to TEA block. In these channels, C‐type inactivation is also unaffected by TEA. 4. These results indicate that the sites where TEA blocks and competes with C‐type inactivation can be segregated. To modulate inactivation, TEA must enter deeply into the channel mouth. These results suggest that C‐type inactivation is not due to a large molecular rearrangement in the outer channel vestibule, but it is essentially produced by a conformational change restricted to a local site in the pore.

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Reduction of Ca2+ channel activity by hypoxia in human and porcine coronary myocytes

OBJECTIVE Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.

Metabotropic Regulation of RhoA/Rho-Associated Kinase by L-type Ca2+ Channels New Mechanism for Depolarization-Evoked Mammalian Arterial Contraction

Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca2+ release). We have studied the functional role of calcium channel-induced Ca2+ release and the inter-relations between Ca2+ channel and RhoA/ROCK activation. Methods and Results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca2+ channel activation and subsequent metabotropic Ca2+ release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca2+ from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca2+ release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca2+ channel activation with metabotropic Ca2+ release and sensitization (RhoA/ROCK stimulation). # Novelty and Significance {#article-title-45}Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca2+ release). We have studied the functional role of calcium channel-induced Ca2+ release and the inter-relations between Ca2+ channel and RhoA/ROCK activation. Methods and Results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca2+ channel activation and subsequent metabotropic Ca2+ release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca2+ from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca2+ release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca2+ channel activation with metabotropic Ca2+ release and sensitization (RhoA/ROCK stimulation).

Metabotropic Regulation of RhoA/Rho-Associated Kinase by L-type Ca2+ Channels

Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca2+ release). We have studied the functional role of calcium channel-induced Ca2+ release and the inter-relations between Ca2+ channel and RhoA/ROCK activation. Methods and Results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca2+ channel activation and subsequent metabotropic Ca2+ release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca2+ from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca2+ release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca2+ channel activation with metabotropic Ca2+ release and sensitization (RhoA/ROCK stimulation). # Novelty and Significance {#article-title-45}Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca2+ release). We have studied the functional role of calcium channel-induced Ca2+ release and the inter-relations between Ca2+ channel and RhoA/ROCK activation. Methods and Results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca2+ channel activation and subsequent metabotropic Ca2+ release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca2+ from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca2+ release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca2+ channel activation with metabotropic Ca2+ release and sensitization (RhoA/ROCK stimulation).

Short Communication: Genetic Ablation of L-Type Ca2+ Channels Abolishes Depolarization-Induced Ca2+ Release in Arterial Smooth Muscle

Rationale: In arterial myocytes, membrane depolarization-induced Ca2+ release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca2+ influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known. Objective: Biophysical and pharmacological data have suggested that L-type Ca2+ channels could be the sensors coupling membrane depolarization to SR Ca2+ release. This hypothesis was tested using smooth muscle–selective conditional Cav1.2 knockout mice. Methods and Results: In aortic myocytes, the decrease of Ca2+ channel density was paralleled by the disappearance of SR Ca2+ release induced by either depolarization or Ca2+ channel agonists. Cav1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca2+ channel–null cells showed unaltered caffeine-induced Ca2+ release and contraction. Conclusion: These data suggest that Cav1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca2+ release. These findings support a novel, ion-independent, functional role of L-type Ca2+ channels linked to intracellular signaling pathways in vascular myocytes.

Hypoxia Inducible Factor-2α Stabilization and Maxi-K+ Channel β1-Subunit Gene Repression by Hypoxia in Cardiac Myocytes: Role in Preconditioning

The Ca2+- and voltage-dependent K+ (maxi-K) channel &bgr;1-subunit mRNA is particularly abundant in cardiomyocytes but its functional role is unknown. This is intriguing because functional maxi-K channels are not found in cardiomyocyte plasmalemma, although they have been suggested to be in the inner mitochondrial membrane and participate in cardioprotection. We report here that &bgr;1 protein may interact with mitochondrial proteins and that the &bgr;1-subunit gene (KCNMB1) is repressed by sustained hypoxia in dispersed cardiomyocytes as well as in heart intact tissue. The effect of hypoxia is time- and dose-dependent, is mimicked by addition of reactive oxygen species, and selectively requires hypoxia inducible factor-2&agr; (Hif-2&agr;) stabilization. We have observed that adaptation to hypoxia exerts a protective role on cardiomyocytes subjected to ischemia and that, unexpectedly, this form of preconditioning absolutely depends on Hif-2&agr;. Interference of the &bgr;1-subunit mRNA increases cardiomyocyte resistance to ischemia. Therefore, Hif-2&agr;-mediated &bgr;1-subunit gene repression is a previously unknown mechanism that could participate in the gene expression program triggered by sustained hypoxia to prevent deleterious mitochondrial depolarization and ATP deficiency in cardiac cells. Our work provides new perspectives for research on cardiac preconditioning.

Prolyl Hydroxylase-dependent Modulation of Eukaryotic Elongation Factor 2 Activity and Protein Translation under Acute Hypoxia

Background: Translational arrest is a classical cellular response to hypoxia, the underlying mechanisms of which are unknown. Results: Inhibitory phosphorylation of eukaryotic elongation factor 2 by acute hypoxia depends on oxygen-sensitive prolyl hydroxylases (PHDs). Conclusion: The elongation phase of protein synthesis is regulated by PHDs. Significance: This work unravels a novel cellular process controlled by PHDs, potential pharmacological targets in several human diseases. Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O2 tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr56 occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.

Homer proteins mediate the interaction between STIM1 and Cav1.2 channels

STIM1 is a ubiquitous Ca2+ sensor of the intracellular, agonist-sensitive, Ca2+ stores that communicates the filling state of the Ca2+ compartments to plasma membrane store-operated Ca2+ (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca2+ influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, β2 and α2δ-1 exhibited a significant Ca2+ entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca2+ stores, resulted in Ca2+ influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 α1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 α1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1.