Intracellular Ca2+ silences L-type Ca2+ channels in mesenteric veins: mechanism of venous smooth muscle resistance to calcium channel blockers.

RATIONALE: Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca(2+) influx through L-type Ca(2+) channels (LTCC) mediates arterial but not venous tone. OBJECTIVE: The object of this study was to resolve the mechanism of venous resistance to CCBs. METHODS AND RESULTS: We compared the sensitivity of depolarization (KCl)-induced constriction of rat small mesenteric arteries (MAs) and veins (MVs) to the dilator effect of CCBs. Initial findings confirmed that nifedipine progressively dilated depolarization-induced constrictions in MAs but not MVs. However, Western blots showed a similar expression of the alpha(1C) pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca(2+) channel current between single smooth muscle cells (SMCs) of MAs and MVs. Based on these findings, we hypothesized that LTCCs are expressed but "silenced" by intracellular Ca(2+) in venous SMCs. After depletion of intracellular Ca(2+) stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca(2+) influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca(2+) stores by caffeine, ryanodine, or 2-aminoethoxydiphenyl borate. Cell-attached patch recordings of unitary LTCC currents confirmed rare channel openings during depolarization of venous compared to arterial SMCs, but chelating intracellular Ca(2+) significantly increased the open-state probability of venous LTCCs. CONCLUSIONS: We report that intracellular Ca(2+) inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained.

Vascular smooth muscle-specific knockdown of the noncardiac form of the L-type calcium channel by microRNA-based short hairpin RNA as a potential antihypertensive therapy.

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (Ca(L)) current and vascular tone is increased because of increased expression of the noncardiac form of the Ca(L) (Ca(v)1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Ca(v)1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotransfected with a rat Ca(v)1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific enhancer/promoter reduced endogenous Ca(v)1.2 expression by 61% and decreased the Ca(L) current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Ca(v)1.2, it did not affect the Ca(L) expression and function in cultured cardiomyocytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Ca(v)1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Ca(v)1.2 siRNA without similarly affecting cardiac Ca(L) expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension.

Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.

Calcium influx through long-lasting ("L-type") Ca(2+) channels (Ca(V)) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca(2+) overload, and interventions that reduce expression of the pore-forming alpha(1) subunit may alleviate cytosolic Ca(2+) excess. As a molecular approach to disrupt the assembly of Ca(V)1.2 (alpha(1C)) channels at the cell membrane, we targeted the Ca(2+) channel beta(2) subunit, an intracellular chaperone that interacts with alpha(1C) via its beta interaction domain (BID) to promote Ca(V)1.2 channel expression. Recombinant adenovirus expressing either the full beta(2) subunit (Full-beta(2)) or truncated beta(2) subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-beta(2) at the surface membrane was associated with increased Ca(2+) current mainly attributed to Ca(V)1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the alpha(1C) protein of Ca(V)1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca(2+) current. C-BID and BID also attenuated Ca(2+) current associated with another L-type Ca(2+) channel, Ca(V)1.3, but they did not reduce transient Ca(2+) currents attributed to Ca(V)3 channels. These results suggest that beta(2) subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca(2+) channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca(2+) overload.

Delivery of ion channel genes to treat cardiovascular diseases.

Modifying ion channel expression and function in the heart and vasculature are potentially useful, novel approaches to managing cardiac hypertrophy, atrial fibrillation and hypertension. Calcium channels play a pivotal role in the heart and vasculature in controlling muscle contraction as well as other aspects of calcium-dependent signaling. The present investigation reports development of mutated L-type calcium channel beta subunits that are delivered by an adenoviral vector to vascular smooth muscle tissue. Wild type subunits serve a chaperone function for the pore-forming alpha(1C) subunit of the calcium channel, localize to the cell membrane and enhance calcium current. Conversely, mutated subunits function as dominant negative, defective chaperone molecules that disrupt targeting to the cell membrane and decrease calcium current. The dominant negative genes can be delivered in vitro and ex vivo, and have the potential to decrease arterial tone and lower blood pressure in vivo.

Early diabetic neuropathy: triggers and mechanisms.

Peripheral neuropathy, and specifically distal peripheral neuropathy (DPN), is one of the most frequent and troublesome complications of diabetes mellitus. It is the major reason for morbidity and mortality among diabetic patients. It is also frequently associated with debilitating pain. Unfortunately, our knowledge of the natural history and pathogenesis of this disease remains limited. For a long time hyperglycemia was viewed as a major, if not the sole factor, responsible for all symptomatic presentations of DPN. Multiple clinical observations and animal studies supported this view. The control of blood glucose as an obligatory step of therapy to delay or reverse DPN is no longer an arguable issue. However, while supporting evidence for the glycemic hypothesis has accumulated, multiple controversies accumulated as well. It is obvious now that DPN cannot be fully understood without considering factors besides hyperglycemia. Some symptoms of DPN may develop with little, if any, correlation with the glycemic status of a patient. It is also clear that identification of these putative non-glycemic mechanisms of DPN is of utmost importance for our understanding of failures with existing treatments and for the development of new approaches for diagnosis and therapy of DPN. In this work we will review the strengths and weaknesses of the glycemic hypothesis, focusing on clinical and animal data and on the pathogenesis of early stages and triggers of DPN other than hyperglycemia.

Neuronal function and alpha3 isoform of the Na/K-ATPase.

The Na/K-ATPase is a complex of integral membrane proteins that carries out active transport of sodium and potassium across the cell plasma membrane, and maintains chemical gradients of these ions. The alpha subunit of the Na/K-ATPase has several isoforms that are expressed in a cell type- and tissue-dependent manner. In adult vertebrates, while kidney cells express mostly alpha1, muscle and glial cells -- alpha1 and alpha2, and sperm cells -- alpha1 and alpha4 isoforms of Na/K-ATPase, neurons may express alpha1, alpha2, alpha3 or any combination of these isoforms, and evidence suggests that neuronal type is the determining factor. The functional significance of multiple isoforms of the Na/K-ATPase and their non-uniform expression, and the link between neuron function and expression of a given isoform of the Na/K-ATPase in particular, remains unknown. Several hypotheses on this account were introduced, and in this work we will review the present status of these hypotheses, and their standing in application to recent data on the expression of isoforms of the Na/K-ATPase in the peripheral nervous system of vertebrate animals.

Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

Mechanical hyperalgesia in rat models of systemic and local hyperglycemia.

Mechanical hyperalgesia is an early symptom of diabetic neuropathy. To evaluate the mechanisms underlying this symptom, it was studied and compared in rat models of systemic and local hyperglycemia. Systemic hyperglycemia was induced by a single injection of streptozotocin (STZ, 50 mg/kg). Local hyperglycemia either in L(5) dorsal root ganglion (DRG) or a segment of the sciatic nerve at mid-thigh level was maintained by perfusion with 30-mM glucose solution delivered from a surgically implanted osmotic minipump. Mechanical hyperalgesia was assessed using modified von Frey filaments and hind limb withdrawal threshold measurements. During 2 weeks of STZ-induced diabetes rat systemic blood glucose level increased from 5.1+/-0.3 to 23+/-1.9 mM and limb withdrawal threshold decreased by approximately 30% bilaterally. During 2 weeks of local perfusion systemic blood glucose did not change; however, rats that underwent perfusion of the DRG or sciatic nerve with glucose exhibited a rapid (completed in approximately 1 week) 40-50% decrease in ipsilateral limb withdrawal threshold. Perfusion of the sciatic nerve with the normoglycemic buffer solution did not affect withdrawal thresholds. The aldose reductase inhibitor sorbinil (2.5 mg/ml) when added to 30-mM glucose perfusion solution prevented hyperalgesia. These data suggest that mechanical hyperalgesia in diabetic animals may, at least in part, result from focal injury caused by a direct toxic effect of glucose in the peripheral nervous system. These data also support the idea of activation of aldose reductase and polyol pathway as an important mechanism of hyperglycemia-induced impairment of nerve function.

Tumor Necrosis Factor-α Suppresses Activation of Sustained Potassium Currents in Rat Small Diameter Sensory Neurons.

Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, produces pain and hyperalgesia by activating and/or sensitizing nociceptive sensory neurons. In the present study, using whole-cell patch clamp techniques, the regulation of potassium currents by TNF-α was examined in acutely dissociated small dorsal root ganglion neurons. We found that acute application of TNF-α inhibited, in a dose-dependent manner, the non-inactivating sustained potassium current without changing the rapidly inactivating transient current or the kinetics of steady-state inactivation. The effects of TNF-α on potassium currents were similar to that of prostaglandin E2 as reported previously and also demonstrated in the current study. Furthermore, indomethacin, a potent inhibitor for both cyclo-oxygenase (COX) -1 and COX-2, completely blocked the effect of TNF-α on potassium currents. These results suggest that TNF-α may sensitize or activate sensory neurons by suppressing the sustained potassium current in nociceptive DRG neurons, possibly via stimulating the synthesis/release of endogenous prostaglandins.

Bradykinin-induced chloride conductance in murine proximal tubule epithelial cells.

Despite the recognized role of bradykinin (BK)-induced calcium and chloride conductance in regulating salt transport in the kidney, the signaling pathway involved has not been well examined. Patch clamp of murine proximal tubule (TKPTS) cells revealed that BK (10 nM) produced an increase in an outwardly rectifying current from a basal level of 2.9 +/- 0.6 to 13.8 +/- 1.1 pA/pF following addition of BK (n = 8; p < 0.001). The shift in reversal potential seen with BK on changing the intracellular solution to 152 mM chloride and significant inhibition of the current by 100 microM 4,4'-di-isothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) suggested that BK activated a chloride current. BK-induced current was blocked by B2 receptor antagonist but not by B1 antagonist or pertussis toxin indicating that the current was mediated by B2 receptors possibly through Gq activation. TMB-8 completely blocked the BK-calcium rise in fura-2 studies but did not block the BK-chloride response indicating that BK-mediated chloride current is calcium-independent. BK-induced current was dependent on phospholipase C (PLC) since U73122, a PLC-beta blocker (10 microM) blocked it completely. Furthermore, chloride conductance was not modulated by bisindolylmaleimide, an inhibitor of protein kinase C (PKC), but was enhanced by dibutyryl cAMP. We conclude that BK-induced rise in chloride current is mediated by B2 receptors and dependent on PLC activation but not dependent on calcium rise. Furthermore, the current can be modulated by cAMP but not PKC.

Differential effects of linoleic Acid metabolites on cardiac sodium current.

9,10-Epoxy-12-octadecenoic acid (EOA), a metabolite of linoleic acid, causes cardiac arrest in dogs. Other metabolites of linoleic acid also have toxic effects. This study investigates the mechanism of action of four of these compounds on cardiac Na(+) current (I(Na)). The whole-cell patch-clamp technique was used to investigate the effects of EOA, 9,10-dihydroxy-12-octadecenoic acid (DHOA), and their corresponding methyl esters (9,10-epoxy-12-octadecenoic methyl ester, EOM; and 9,10-dihydroxy-12-octadecenoic methyl ester, DHOM) on I(Na) in isolated adult rat ventricular myocytes. Extracellular application of each compound elicited a concentration-dependent inhibition of I(Na). The dose-response curve yielded 50% inhibition concentrations of 301 +/- 117 microM for DHOA, 41 +/- 6 microM for DHOM, 34 +/- 5 microM for EOA, and 160 +/- 41 microM for EOM. Although there was no effect on activation, 50 microM DHOM, EOA, and EOM significantly hyperpolarized the steady-state inactivation curve by approximately -6 mV. Furthermore, EOM significantly increased the slope of the steady-state inactivation curve. These compounds also seemed to stabilize the inactivated state because the time for recovery from inactivation was significantly slowed from a control value of 12.9 +/- 0.5 ms to 30.5 +/- 3.3, 31.4 +/- 1.4, and 20.5 +/- 1.0 ms by 50 microM DHOM, EOA, and EOM, respectively. These compounds have multiple actions on Na(+) channels and that despite their structural similarities their actions differ from each other. The steady-state block of I(Na) suggests that either the pore is being blocked or the channels are prevented from gating to the open state. In addition, these compounds stabilize the inactivated state and promote increased population of a slower inactivated state.

Relevance of hyperglycemia to early mechanical hyperalgesia in streptozotocin-induced diabetes.

A modified von Frey filament test and an algesiometer paw pressure test were used to measure mechanical nociceptive withdrawal thresholds of the hind limb of control rats and rats injected with streptozotocin (STZ, 50 mg/kg). STZ treatment induced hyperglycemia (HG rats) in about 40% of treated animals. The rest of the STZ-treated and control rats remained normoglycemic (NG rats) throughout the entire experiment. No indications of mechanical hyperalgesia were observed in control groups of animals injected with physiological buffer only. However, both the behavioral tests used detected a 15-30% decrease in the mechanical nociceptive threshold of rats treated with STZ. Furthermore, mechanical nociceptive threshold changes were statistically indistinguishable between NG and HG rats. Glucose tolerance test did not reveal abnormalities of glucose metabolism in NG rats (compared to control animals). However, 1 week after STZ injection, the serum insulin level of NG rats was significantly lower than that of age-matched control rats (0.81 +/- 0.16 vs. 3.5 +/- 0.4 ng/mL; p < 0.01). These data strongly argue that systemic hyperglycemia is not the only factor triggering the development of mechanical hyperalgesia in the STZ rat model of diabetes. Other than hyperglycemia, consequences of insulinemia or insulinemia itself may play an important role in early impairment of mechanical nociception in this animal model.

Effect of linoleic acid metabolites on Na(+)/K(+) pump current in N20.1 oligodendrocytes: role of membrane fluidity.

Metabolic derivatives of linoleic acid, both monoepoxides and diols, have been reported to be toxic in humans and multiple animal tissue preparations. A previous electrophysiological study has shown these compounds produce multiple effects on the electrical activity of rat ventricular myocytes. The hydrophobic nature of these compounds suggests the possibility that these effects may be due to nonspecific lipid interactions, i.e., changes in membrane fluidity. This study investigates membrane fluidity as a possible mechanism by which linoleic acid metabolites inhibit Na(+)/K(+) pump current (I(p)). This study showed that positional isomers 9,10- and 12,13-epoxy-octadecenoic acid (EOA) and 9,10- and 12,13-dihydroxy-OA (DHOA) inhibit I(p) in a dose-dependent manner in N20.1 mouse oligodendrocytes, with greater inhibition produced by EOAs. These compounds, at 10 microM, inhibited I(p) by 4.7 +/- 1.6, 18.2 +/- 0.5, 11.7 +/- 0.5, and 25.1 +/- 0.9% for 12,13-DHOA, 9,10-DHOA, 12,13-EOA, and 9,10-EOA, respectively, in oligodendrocytes. Fluorescence recovery after photobleaching measurements showed that both DHOA isomers produced a 7-8% increase in diffusion coefficient of the probe at 10 microM, whereas the diffusion coefficient was decreased by 5 and 13% by 9,10-EOA and 12,13-EOA, respectively. There was no apparent correlation between membrane fluidity and inhibition of I(p) by these four linoleic acid metabolites. These results indicate that membrane fluidity alone cannot explain the effects of these compounds on I(p) and suggest that they have a specific interaction with the Na(+)/K(+) pump.