bluuebunny/crossref_metadata_2025 huggingface dataset:
Negative Chronotropic Effect of Endothelin 1 Mediated Through ET
A
Receptors in Guinea Pig Atria
Authors: Ono Kageyoshi , Eto Koji , Sakamoto Aiji , Masaki Tomoh , Shibata Katsushi , Sada Toshio , Hashimoto Keitaro , Tsujimoto Gozoh
Published: June 2012

Abstract
Endothelins exert potent excitatory cardiac effects by acting on specific receptors on myocytes. In this study, we have examined the signal transduction mechanism for the chronotropic effect of endothelins in guinea pig atria. A competition binding of [
125
I]endothelin 1 ([
125
I]ET-1) using the recently developed ET
A
receptor–selective antagonist BQ123 showed the presence of almost equal populations of ET
A
(44%) and ET
B
(56%) receptors in the guinea pig right atria. In a concentration-response study, endothelin 3 (ET-3), an agonist with higher affinity to ET
B
receptors than to ET
A
receptors, and sarafotoxin S6c (STXS6c), an ET
B
receptor–selective agonist, increased the rate of spontaneous beating at all concentrations tested (10 pmol/L to 100 nmol/L). In contrast, ET-1, a nonselective agonist, increased the heart rate at lower concentrations (10 pmol/L to 10 nmol/L) but decreased it at higher concentrations (30 to 100 nmol/L). When ET-1 (100 nmol/L) was applied in a single amount, heart rate was strongly increased; however, this increase was followed by a rapid decline in the response. ET-1 (100 nmol/L) but not ET-3 or STXS6c significantly reduced the heart rate when it was raised by isoproterenol (ISO, 300 nmol/L) either in the absence or presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Correspondingly, ET-1 significantly reduced the ISO-induced elevation of cAMP accumulation (19.1±1.7 pmol/mg protein [n=8] and 12.6±1.2 pmol/mg protein [n=7] in the absence and presence of ET-1, respectively;
P
<.01), which was also observed even in the presence of IBMX. Treatment with BQ123 (1 μmol/L) abolished these inhibitory effects of ET-1 on both the chronotropy and the cAMP accumulation. Also, pretreatment of guinea pigs with pertussis toxin (5 μg/100 g body wt IV) abolished the inhibitory effects of ET-1. These data showed that ET
A
receptors are involved in an inhibitory cardiac action of endothelins, which is coupled to a pertussis toxin–sensitive G protein/adenylate cyclase inhibition pathway. This ET
A
receptor–mediated inhibitory action gives new insights into understanding physiological and pathophysiological modulations of cardiac functions by endothelins.
Inhibition of Vascular Smooth Muscle Cell K
+
Currents by Tyrosine Kinase Inhibitors Genistein and ST 638
Authors: Smirnov Sergey V. , Aaronson Philip I.
Published: June 2012

Abstract
The whole-cell patch-clamp technique was used to characterize the effects of several tyrosine kinase inhibitors on the voltage-gated K
+
current (I
K
) in rat and rabbit pulmonary artery cells. I
K
was blocked in a dose-dependent manner by genistein (20 to 100 μmol/L) and ST 638 (0.5 to 40 μmol/L) but not by the inactive genistein analogue diadzein (100 μmol/L). This inhibition was not significantly altered when ATP was excluded from the patch pipette or when it was replaced by the poor tyrosine kinase substrate ATP-γ-S. The inhibition was also unaffected by inclusion of the tyrosine phosphatase inhibitor orthovanadate in either the bath (0.5 mmol/L) or pipette (0.2 mmol/L) solutions. In the rat, I
K
ordinarily inactivated negligibly over 300 ms. In the presence of 10 μmol/L ST 638, however, I
K
reached a peak ≈5 ms after depolarization (to +60 mV) and then decayed markedly. In the rabbit, I
K
demonstrated a prominent rapidly decaying initial component that was only slightly inhibited by ST 638, which preferentially blocked the sustained current; genistein showed the opposite selectivity. These observations indicated that I
K
blockade by genistein and ST 638 was not mediated by an inhibition of tyrosine kinase activity and further suggested that in both types of cells genistein and ST 638 preferentially blocked rapidly and slowly inactivating components of I
K
, respectively.
Unitary Chloride Channels Activated by Protein Kinase C in Guinea Pig Ventricular Myocytes
Authors: Collier Mei Lin , Hume Joseph R.
Published: June 2012

Abstract
Recent evidence suggests that protein kinase A (PKA)-activated Cl
−
channels in heart are encoded by an isoform of the epithelial cystic fibrosis transmembrane conductance regulator gene (CFTR). Macroscopic current measurements indicate that a similar time-independent Cl
−
conductance can be activated through a protein kinase C (PKC)-dependent pathway in guinea pig and feline ventricle. However, it is presently not clear whether PKC is activating the same population of channels as PKA or a separate class of Cl
−
channels, even though the regulatory (R) domain of CFTR is known to contain consensus phosphorylation sites for both PKA and PKC. In the present study we directly compare the properties of single Cl
−
channels activated by PKC and PKA in cell-attached patches of guinea pig ventricular myocytes. Pipette and bath solutions contained
N
-methyl-
d
-glucamine and Cs
+
or tetraethylammonium as substitutes for Na
+
and K
+
, respectively, and Cl
−
concentration in the patch pipette was either 150 mmol/L or 40 mmol/L. Bath application of phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate (PDBu or PMA; 50 nmol/L), activators of PKC, resulted in the appearance of unitary Cl
−
channels with a mean conductance of 9.31±0.94 pS (n=8) and 8.8 pS (n=2), respectively, and reversal potentials were close to predicted E
Cl
. Addition of staurosporine (500 nmol/L) reduced open probability (P
o
) of channels activated by PDBu. Bath application of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 μmol/L) resulted in the activation of Cl
−
channels with a conductance (mean 8.76±0.67 pS, n=3) similar to those activated by PDBu. Open probability (P
o
) of both PKC- and PKA-activated Cl
−
channels exhibited voltage independence and both were insensitive to block by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). In patches containing channels preactivated by PDBu, subsequent bath application of IBMX (500 μmol/L) resulted in an increase in P
o
without any evidence for the activation of a separate population of channels with a different unitary conductance. These results suggest either that PKC can activate cardiac CFTR Cl
−
channels and that once activated by PKC, the channels can be further modulated by PKA or that PKC and PKA activate separate populations of Cl
−
channels with similar conductance and kinetic properties.
Functional Consequences of Sulfhydryl Modification in the Pore-Forming Subunits of Cardiovascular Ca
2+
and Na
+
Channels
Authors: Chiamvimonvat Nipavan , O’Rourke Brian , Kamp Timothy J. , Kallen Roland G. , Hofmann Franz , Flockerzi Veit , Marban Eduardo
Published: June 2012

Abstract
The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryl-oxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca
2+
channel and the human cardiac tetrodotoxin-insensitive Na
+
channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca
2+
or Na
+
channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca
2+
and Na
+
channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2′-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca
2+
current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca
2+
-channel specific: DTDP induced no changes in expressed human cardiac Na
+
current. Single-channel Ba
2+
current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca
2+
current reflects changes in gating and not permeation. In summary, the pore-forming (α
1
) subunit of the L-type Ca
2+
channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.
Role of Lipoxygenase Metabolites in Ischemic Preconditioning
Authors: Murphy Elizabeth , Glasgow Wayne , Fralix Teresa , Steenbergen Charles
Published: June 2012

Abstract
Preconditioning with brief intermittent periods of ischemia before a sustained period of ischemia has been shown to reduce infarct size and improve recovery of function in rat hearts. The mediators of this protective response are unknown in rats. We tested the hypothesis that a lipoxygenase metabolite might be involved in preconditioning, since lipoxygenase metabolites such as 12-hydroperoxyeicosatetraenoic acid have been shown to increase K
+
channel activity and to decrease Ca
2+
channel activity, which could have a protective effect on ischemic injury. In support of this hypothesis, we report that the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA, 5 μmol/L) and eicosatetraynoic acid (7 μmol/L) added just before and during preconditioning blocked the protective effects of preconditioning on recovery of function during reflow after 30 minutes of global ischemia. In addition, these lipoxygenase inhibitors partially blocked the ability of preconditioning to attenuate the rise in cytosolic free calcium during sustained ischemia. We also investigated the effects of preconditioning on eicosanoid metabolism by using high-performance liquid chromatography and found that 12-hydroxyeicosatetraenoic acid (12-HETE), the stable product of the lipoxygenase pathway, was made during the preconditioning protocol and that 12-HETE accumulation was blocked by NDGA. Thus, there is a correlation between functional recovery after ischemia and stimulation of the lipoxygenase pathway of arachidonic acid metabolism before the sustained period of ischemia; inhibition of the lipoxygenase pathway eliminates the protective effect of preconditioning on recovery of function after ischemia.
Mechanism of Impaired Myocardial Function During Progressive Coronary Stenosis in Conscious Pigs
Authors: Shen You-Tang , Vatner Stephen F.
Published: June 2012

Abstract
The major goal of this study was to determine whether impaired myocardial contractile function during the development of progressive coronary artery stenosis induced by ameroid constriction in conscious pigs reflected myocardial “hibernation” or “stunning.” Minipigs were instrumented with a coronary ameroid constrictor and hydraulic occluder, regional wall thickness crystals, a left ventricular (LV) pressure gauge, and aortic and left atrial catheters. In the seven pigs in which it was measured, systolic wall thickening (WT) distal to the ameroid fell by a maximum of 56±6% at 20±3 days after ameroid implantation and then began to recover. At 1 day after ameroid implantation, brief complete coronary artery occlusion (CAO) resulted in wall thinning distal to the ameroid (−113±4%) and transmural decreases in myocardial blood flow in endocardial (from 0.82±0.08 to 0.02±0.01 mL/min per gram) and epicardial (from 0.73±0.13 to 0.03±0.02 mL/min per gram) layers. At 20±3 days, baseline myocardial blood flow was not altered either in endocardial (0.92±0.10 mL/min per gram) or epicardial (0.85±0.12 mL/min per gram) layers, whereas brief complete coronary artery occlusion still reduced WT (−83±12%) and myocardial blood flow in endocardial (to 0.21±0.03 mL/min per gram) and epicardial (to 0.43±0.12 mL/min per gram) layers, indicating that the coronary artery was not totally occluded. Pathology in four pigs demonstrated no gross necrotic myocardium shortly after this time point. Transient reductions in WT distal to the ameroid were observed during progressive coronary artery stenosis in response to spontaneous increases in activity. Beat-by-beat analysis of these episodes revealed that acute reductions in WT followed increases in LV dP/dt and heart rate and exhibited delayed recovery. These data suggest that the reduced function during ameroid-induced coronary stenosis reflected cumulative myocardial stunning rather than a primary deficit in coronary blood flow or “hibernating myocardium.”
Exercise Training Augments Flow-Dependent Dilation in Rat Skeletal Muscle Arterioles
Authors: Koller Akos , Huang An , Sun Dong , Kaley Gabor
Published: June 2012

Abstract
We aimed to test the hypothesis that as a consequence of short-term daily exercise, flow (shear stress)–dependent dilation and its mediation by the endothelium are altered in skeletal muscle arterioles. After initial familiarization with the protocol, rats ran on a treadmill once a day (with gradually increasing intensity up to 40 minutes and 28 m/min) for ≈3 weeks (EX group); a control group remained sedentary (SED group). The active (internal) diameters of isolated gracilis muscle arterioles of SED and EX rats at 80 mm Hg were significantly different (55.2±2.1 and 49.3±2.0 μm,
P
<.05), and their passive diameters (in Ca
2+
-free solution) were 105.3±3.1 and 111.2±2.4 μm (not significantly different), respectively. Increases in flow of the perfusion solution from 0 to 12 μL/min elicited a significantly greater increase in diameter of EX arterioles (by 83.5% at maximum flow). This enhanced sensitivity maintained a lower shear stress in EX arterioles (15 to 20 dyne/cm
2
) compared with SED arterioles (25 to 35 dyne/cm
2
). In both SED and EX arterioles, flow-dependent dilation was eliminated after removal of the endothelium. Either
N
ω
-nitro-
l
-arginine, a nitric oxide synthase inhibitor, or indomethacin, an inhibitor of prostaglandin synthesis, shifted the flow-diameter and calculated wall shear stress–diameter curves significantly to the right. Each of the inhibitors reduced flow-dependent dilation to a similar degree (≈40% to 45%); their combined administration nearly completely eliminated the dilation of arterioles of both SED and EX rats. Thus, we conclude that the sensitivity of gracilis muscle arterioles of rats to wall shear stress is upregulated after short-term daily exercise, resulting in an augmented dilator response that is due to an increased release of both endothelium-derived nitric oxide and prostaglandins.
Arginine Vasopressin–Induced Potentiation of Unitary L-Type Ca
2+
Channel Current in Guinea Pig Ventricular Myocytes
Authors: Zhang Shetuan , Hirano Yuji , Hiraoka Masayasu
Published: June 2012

Abstract
The effects of arginine vasopressin (AVP) on L-type Ca
2+
channels were studied by recording single-channel activity from cell-attached patches on isolated guinea pig ventricular myocytes, with 100 mmol/L Ba
2+
used as the charge carrier. Bath application of AVP (100 nmol/L) reversibly increased channel open probability by a factor of 2.92±1.43 (n=15) because of the increased number of channel openings and increased open times. AVP did not change the amplitudes of single-channel currents (1.17±0.10 pA in the control condition and 1.12±0.11 pA after AVP, at +20 mV; n=6). In our experimental conditions, in which myocytes were bathed in Ca
2+
-free high-potassium solutions, AVP-induced potentiation was observed without changes in [Ca
2+
]
i
measured by fura 2 fluorescence signals (estimated [Ca
2+
]
i
, ≈80 nmol/L). The AVP-induced increase in channel open probability was abolished by OPC-21268 (8 μmol/L), a specific blocker of V
1
receptor, but not by a V
2
blocker, OPC-31260 (5 μmol/L). AVP-induced potentiation was also suppressed by a broad-spectrum protein kinase inhibitor, H7 (100 μmol/L, bath application), but not by H89 (1 μmol/L), a blocker with high specificity to protein kinase A. AVP application after the treatment by phorbol ester (phorbol 12-myristate 13-acetate, 100 nmol/L for 1 hour) failed to potentiate the channel activity. These results raised the possibility that protein kinase C might be involved during signal transduction. Our results provide direct evidence that AVP potentiates cardiac L-type Ca
2+
currents via V
1
receptor stimulation.
Myosin Light Chain–Actin Interaction Regulates Cardiac Contractility
Authors: Morano Ingo , Ritter Oliver , Bonz Andreas , Timek Tomasz , Vahl Christian F. , Michel Gerd
Published: June 2012

Abstract
The amino-terminal domain of the essential myosin light chain (MLC-1) binds to the carboxy terminus of the actin molecule. We studied the functional role of this interaction by two approaches: first, incubation of intact and chemically skinned human heart fibers with synthetic peptide corresponding to the sequences 5 through 14 (P5-14), 5 through 8 (P5-8), and 5 through 10 (P5-10) of the human ventricular MLC-1 (VLC-1) to saturate actin-binding sites, and second, incubation of skinned human heart fibers with a monoclonal antibody (MabVLC-1) raised against the actin-interacting N-terminal domain of human VLC-1 using P5-14 as antigen to deteriorate VLC-1 binding to actin. P5-14 increased isometric tension generation of skinned human heart fibers at both submaximal and maximal Ca
2+
activation, the maximal effective peptide dosage being in the nanomolar range. A scrambled peptide of P5-14 with random sequence had no effects up to 10
−8
mol/L, ie, where P5-14 was maximally effective. P5-8 and P5-10 increased isometric force to the same extent as P5-14, but micromolar concentrations were required. Amplitude of isometric twitch contraction, rate of tension development, rate of relaxation, and shortening velocity at near-zero load of electrically driven intact human atrial fibers increased significantly on incubation with P5-14. These alterations were not associated with modulation of intracellular Ca
2+
transients as monitored by fura 2 fluorescence measurements. Incubation of skinned human heart fibers with MabVLC-1 increased isometric tension at both submaximal and maximal Ca
2+
activation levels, having a maximal effective concentration in the femtomolar range. We conclude that VLC-1–actin interaction modulates cardiac contractility and may be a target for new inotropic intervention.
Effects of Action Potential Duration on Excitation-Contraction Coupling in Rat Ventricular Myocytes
Authors: Bouchard R. A. , Clark R. B. , Giles W. R.
Published: June 2012

Abstract
Although each of the fundamental processes involved in excitation-contraction coupling in mammalian heart has been identified, many quantitative details remain unclear. The initial goal of our experiments was to measure both the transmembrane Ca
2+
current, which triggers contraction, and the Ca
2+
extrusion due to Na
+
-Ca
2+
exchange in a single ventricular myocyte. An action potential waveform was used as the command for the voltage-clamp circuit, and the membrane potential, membrane current, [Ca
2+
]
i
, and contraction (unloaded cell shortening) were monitored simultaneously. Ca
2+
-dependent membrane current during an action potential consists of two components: (1) Ca
2+
influx through L-type Ca
2+
channels (I
Ca-L
) during the plateau of the action potential and (2) a slow inward tail current that develops during repolarization negative to ≈−25 mV and continues during diastole. This slow inward tail current can be abolished completely by replacement of extracellular Na
+
with Li
+
, suggesting that it is due to electrogenic Na
+
-Ca
2+
exchange. In agreement with this, the net charge movement corresponding to the inward component of the Ca
2+
-dependent current (I
Ca-L
) was approximately twice that during the slow inward tail current, a finding that is predicted by a scheme in which the Ca
2+
that enters during I
Ca
is extruded during diastole by a 3 Na
+
–1 Ca
2+
electrogenic exchanger. Action potential duration is known to be a significant inotropic variable, but the quantitative relation between changes in Ca
2+
current, action potential duration, and developed tension has not been described in a single myocyte. We used the action potential voltage-clamp technique on ventricular myocytes loaded with indo 1 or rhod 2, both Ca
2+
indicators, to study the relation between action potential duration, I
Ca-L
, and cell shortening (inotropic effect). A rapid change from a “short” to a “long” action potential command waveform resulted in an immediate decrease in peak I
Ca-L
and a marked slowing of its decline (inactivation). Prolongation of the action potential also resulted in slowly developing increases in the magnitude of Ca
2+
transients (145±2%) and unloaded cell shortening (4.0±0.4 to 7.6±0.4 μm). The time-dependent nature of these effects suggests that a change in Ca
2+
content (loading) of the sarcoplasmic reticulum is responsible. Measurement of [Ca
2+
]
i
by use of rhod 2 showed that changes in the rate of rise of the [Ca
2+
]
i
transient (which in rat ventricle is due to the rate of Ca
2+
release from the sarcoplasmic reticulum) were closely correlated with changes in the magnitude and the time course of I
Ca-L
. These findings demonstrate that Ca
2+
release from the sarcoplasmic reticulum can be modulated by the action potential waveform as a result of changes in I
Ca-L
.
Spatial Distribution of Connexin43, the Major Cardiac Gap Junction Protein, Visualizes the Cellular Network for Impulse Propagation From Sinoatrial Node to Atrium
Authors: Velde I. ten , de Jonge B. , Verheijck E.E. , van Kempen M.J.A. , Analbers L. , Gros D. , Jongsma H.J.
Published: June 2012

Abstract
Myocytes are electrically coupled by gap junctions, which are composed of low-resistance intercellular channels. The major cardiac gap junction protein is connexin43 (Cx43). The distribution of Cx43 has been studied by immunofluorescence to visualize the electrical coupling between atrial tissue and sinoatrial node. From modeling studies, this coupling was inferred to be gradual in order to shield the sinoatrial node from the atrial hyperpolarizing influence. The actual Cx43 labeling pattern did not show the expected gradient but instead a rather black and white staining in a striking pattern of strands of cells. We used an immunohistochemical marker (anti–α-smooth muscle actin [αSMA]) that specifically cross-reacts with guinea pig sinoatrial node cells together with Cx43 antibody to stain previously electrophysiologically mapped sinoatrial nodes. We found that in the guinea pig sinoatrial node the impulse originates in an αSMA-positive, virtually Cx43-negative, region (primary pacemaker region). The impulse then travels obliquely upward to the crista terminalis through a region where layers of αSMA-positive cells alternate with layers of Cx43-positive SMA-negative cells. The layers of Cx43-positive cells appear to become broader and thicker in the direction of the crista terminalis, whereas the layers of αSMA-positive cells become thinner and narrower. Lateral contacts between Cx43- and αSMA-positive cells were very sparse and only detected where the Cx43-positive strands ended (the region where αSMA-positive cells fill the whole space between endocardium and epicardium, ie, the putative primary pacemaker region). From these results, we conclude that the primary pacemaker is shielded from the hyperpolarizing influence of the atrium by a gradient in coupling brought about by tissue geometric factors rather than by a gradient of gap junction density.
Oxygen-Derived Free Radical Stress Activates Nonselective Cation Current in Guinea Pig Ventricular Myocytes
Authors: Jabr Rita I. , Cole William C.
Published: June 2012

Abstract
Oxygen-derived free radicals (O-Rs) cause alterations in cardiac electrical activity, including sustained depolarization, which may contribute to arrhythmic activity in reperfusion after ischemia. The ionic current(s) and cellular mechanism(s) underlying the sustained depolarization are not well defined. We used the whole-cell variant of the patch-clamp technique to study sustained depolarization in guinea pig ventricular myocytes during the extracellular application of O-Rs (generating system: dihydroxyfumaric acid, 3 to 6 mmol/L; FeCl
3
/ADP, 0.05:0.5 mmol/L). Myocytes superfused with O-Rs (pipette EGTA, 0.1 mmol/L) showed (1) sustained depolarization to between −40 and −10 mV, (2) oscillations in membrane potential, and (3) triggered activity. The depolarization resulted from an increase in quasi–steady state difference current reversing at ≈−18 mV, and the oscillations were due to transient inward current. The latter were inhibited with ryanodine (10 μmol/L) or high pipette EGTA (5 mmol/L), but the steady state current was unaffected. Nonselective cation current (I
NSC
) (recorded with Cs
+
, Li
+
, and Mg
2+
replacing K
+
, Na
+
, and Ca
2+
, respectively; 20 mmol/L tetraethylammonium chloride [TEA] and 5 mmol/L BAPTA in the pipette solution and 10 mmol/L TEA, 10 μmol/L tetrodotoxin, and 10 μmol/L nicardipine in the bath solution) was activated by O-Rs; the increase in current was unaffected by preventing changes in [Ca
2+
]
i
but was inhibited with dithiothreitol. Oxidizing agents (diamide and thimerosal) or caffeine (pipette EGTA, 0.1 mmol/L) produced a similar increase in membrane conductance. I
NSC
activated with O-Rs, oxidizing agents, or caffeine was sensitive to SK&F 96365. O-R treatment was without effect when I
NSC
was already activated with caffeine. The data suggest that (1) extracellular O-Rs activate a Ca
2+
-sensitive I
NSC
in the absence of changes in [Ca
2+
]
i
, (2) oxidative modification of extracellular sulfhydryl groups may be involved, and (3) this mechanism is different from the Ca
2+
-dependent activation of I
NSC
by intracellular O-Rs, indicating that O-Rs may alter ion channel activity by differential mechanisms, depending on the compartment, extracellular or intracellular, in which they are present.
Biphasic Restitution of Action Potential Duration and Complex Dynamics in Ventricular Myocardium
Authors: Watanabe Mari , Otani Niels F. , Gilmour Robert F.
Published: June 2012

Abstract
The purpose of this study was to determine whether biphasic restitution of action potential duration (APD) in ventricular muscle permits the development of complex dynamic behavior. Such behavior is expected because of the steep ascending slope of restitution and the presence of a maximum. Action potentials recorded from strips of epicardial muscle in which biphasic APD restitution occurred demonstrated a characteristic pattern of phase locking during progressive shortening of the pacing cycle length. 1:1 locking was replaced by irregular dynamics, which in turn was replaced by higher order periodic behavior (eg, 8:8 locking), then by 2:2 locking, and finally by 2:1 locking. Similar patterns of dynamic behavior were produced in a computer model by using a piecewise linear approximation of biphasic APD restitution. Features of APD restitution that were critical determinants of irregular dynamics included the slopes of the ascending and the nonmonotonic regions. These results suggest that rate-related alterations of APD and refractoriness may be affected significantly by small nonmonotonicities in APD restitution.
Dilatation of Cerebral Arterioles in Response to Activation of Adenylate Cyclase Is Dependent on Activation of Ca
2+
-Dependent K
+
Channels
Authors: Taguchi Hisao , Heistad Donald D. , Kitazono Takanari , Faraci Frank M.
Published: June 2012

Abstract
The role of Ca
2+
-dependent potassium channels in mediating vascular responses to activation of adenylate cyclase in vivo is not known. The goal of this study was to examine the hypothesis that dilatation of cerebral arterioles in response to activation of adenylate cyclase is mediated by activation of Ca
2+
-dependent potassium channels. Diameters of cerebral arterioles were measured in vivo in anesthetized rabbits. Topical application of forskolin (1 and 10 μmol/L), a direct activator of adenylate cyclase, dilated cerebral arterioles by 40±8% (mean±SEM) and 71±9%, respectively, from a control diameter of 85±4 μm. Iberiotoxin (50 and 100 nmol/L), a selective inhibitor of Ca
2+
-dependent potassium channels, inhibited dilatation in response to both concentrations of forskolin by 45% to 60%. We obtained similar results by using charybdotoxin (50 nmol/L), another inhibitor of Ca
2+
-dependent potassium channels. Vasodilatation in response to dibutyryl cAMP (a cell-permeable cAMP analogue) was also inhibited by iberiotoxin. In contrast, dilatation of cerebral arterioles in response to sodium nitroprusside and acetylcholine (activators of guanylate cyclase) and aprikalim (activator of ATP-sensitive potassium channels) was not inhibited by iberiotoxin. These findings suggest that dilatation of cerebral arterioles in response to forskolin and increases in intracellular concentrations of cAMP are mediated by activation of Ca
2+
-dependent potassium channels. Thus, activation of Ca
2+
-dependent potassium channels may be a major mechanism of cerebral vasodilatation in response to activation of adenylate cyclase in vivo.
A Subpopulation of Smooth Muscle Cells in Injured Rat Arteries Expresses Platelet-Derived Growth Factor–B Chain mRNA
Authors: Lindner Volkhard , Giachelli Cecilia M. , Schwartz Stephen M. , Reidy Michael A.
Published: June 2012

Abstract
Proliferation of smooth muscle cells (SMCs) and formation of a neointima are characteristics of the response of rat carotid arteries to balloon injury. Rat platelet-derived growth factor (PDGF)-B was cloned, thus allowing us to use species-specific probes to carry out in situ hybridization on the surface of injured arteries. A distinct population of luminal SMCs (7% to 10%) in the developing neointima expressed PDGF-B mRNA, but very few luminal SMCs still expressed PDGF-B (0.5%) when the lesion had stopped growing. Primary SMC cultures revealed expression of PDGF-B mRNA in 1.6% of SMCs derived from normal tunica media and in 11% of SMCs derived from the neointima. These data demonstrate that SMCs in the injured vessel wall are heterogeneous with regard to PDGF-B expression and that subculturing of these cells may give rise to cultures that are either positive or negative for PDGF-B expression. Furthermore, with abundant expression of the PDGF receptor β-subunit expressed by intimal SMCs, our findings provide evidence that PDGF-B synthesized by these cells may be involved in intimal lesion formation via a paracrine or autocrine mechanism.
[Ca
2+
]
i
Inhibition of K
+
Channels in Canine Renal Artery
Authors: Gelband Craig H. , Hume Joseph R.
Published: June 2012

Abstract
The patch-clamp technique was used to examine the inhibition of delayed rectifier K
+
channels by agents that release intracellular Ca
2+
. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 μmol/L) present to inhibit delayed rectifier K
+
current (I
K(dr)
) and Ca
2+
-activated Cl
−
current (I
Cl(Ca)
), angiotensin II (Ang II) and caffeine increased Ca
2+
-activated K
+
current (I
K(Ca)
) between −25 and 80 mV (n=5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 μmol/L) present to inhibit I
K(Ca)
and I
Cl(Ca)
, Ang II and caffeine only caused inhibition of I
K(dr)
. Block was achieved within 15 seconds of drug application and was reversible upon washout (n=5). The effects of Ang II on I
K(Ca)
and I
K(dr)
were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n=3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both I
K(Ca)
and I
K(dr)
. In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 μmol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26±2.9 mV depolarization from −54±3.1 to −28±1.7 mV (n=6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca
2+
indicator indo 1 (100 μmol/L), Ang II, caffeine, and 4-AP increased [Ca
2+
]
i
and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca
2+
]
i
that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca
2+
]
i
(n=4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca
2+
-activated K
+
channels, a 45±7-picosiemen 4-AP–sensitive K
+
channel was identified that was sensitive to cytoplasmic Ca
2+
(n=6). Increasing intracellular Ca
2+
decreased channel opening probability [N×P(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca
2+
from <5 to 200 and 600 nmol/L free Ca
2+
decreased N×P(open) by 52±3% and 73±7%, respectively (n=6). The decrease in opening probability of the delayed rectifier K
+
channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively. These results suggest that agonist-induced changes in intracellular Ca
2+
can alter vascular smooth muscle membrane potential through regulation of delayed rectifier K
+
channels.
Two Components of the Delayed Rectifier K
+
Current in Ventricular Myocytes of the Guinea Pig Type
Authors: Zeng Jinglin , Laurita Kenneth R. , Rosenbaum David S. , Rudy Yoram
Published: June 2012

Abstract
Two distinct delayed rectifier K
+
currents, I
Kr
and I
Ks
, were found recently in ventricular cells. We formulated these currents theoretically and investigated their roles in action potential repolarization and the restitution of action potential duration (APD). The Luo-Rudy (L-R) model of the ventricular action potential was used in the simulations. The single delayed rectifier K
+
current in the model was replaced by I
Kr
and I
Ks
. Our results show that I
Ks
is the major outward current during the plateau repolarization. A specific block of either I
Kr
or I
Ks
can effectively prolong APD to the same degree. Therefore, either channel provides a target for class III antiarrhythmic drugs. In the simulated guinea pig ventricular cell, complete block of I
Kr
does not result in early afterdepolarizations (EADs). In contrast, >80% block of I
Ks
results in abnormal repolarization and EADs. This behavior reflects the high I
Ks
-to-I
Kr
density ratio (≈8:1) in this cell and can be reversed (ie, I
Kr
block can cause EADs) by reducing the ratio of I
ks
to I
Kr
. The computed APD restitution curve is consistent with the experimental behavior, displaying fast APD variation at short diastolic intervals (DIs) and downward shift at longer DIs with the decrease of basic drive cycle length (BCL). Examining the ionic currents and their underlying kinetic processes, we found that activation of both I
Kr
and I
Ks
is the primary determinant of the APD restitution at shorter DIs, with Ca
2+
current through L-type channels (I
Ca
) playing a minor role. The rate of APD change depends on the relative densities of I
Kr
and I
Ks
; it increases when the I
Kr
-to-I
Ks
density ratio is large. The BCL-dependent shift of restitution at longer DIs is primarily attributed to long-lasting changes in [Ca
2+
]
i
. This in turn causes different degrees of Ca
2+
-dependent inactivation of I
Ca
and different degrees of Ca
2+
-dependent conductance of I
Ks
at very long DIs (>5 s) for different BCLs. This BCL dependence of I
Ca
and I
Ks
that is secondary to long-lasting changes in [Ca
2+
]
i
is responsible for APD changes at long DIs and can be viewed as a “memory property” of cardiac cells.
Binding of A
1
Adenosine Receptor Ligand [
3
H]8-Cyclopentyl-1,3-Dipropylxanthine in Coronary Smooth Muscle
Authors: Hussain Tahir , Mustafa S. Jamal
Published: June 2012

Abstract
Vascular smooth muscle has been reported to contain the A
1
subtype of adenosine receptors, but the existence of such receptor(s) in coronary smooth muscle has not been established. In the present study, the
3
H-labeled A
1
-selective antagonist [
3
H]8-cyclopentyl-1,3-dipropylxanthine ([
3
H]DPCPX) was used to demonstrate the specific binding in porcine coronary artery smooth muscle membranes. The binding was saturable with a B
max
of 6.43±1.02 fmol/mg protein. Scatchard analysis of the binding data provided a single binding site with a
K
d
of 0.21±0.025 nmol/L. In the competition experiments, adenosine receptor agonists and antagonists showed the following order of potency (nmol/L):
S
-
N
6
-(2-endo-norbornyl)adenosine (S-ENBA) 0.11=
R
(−)-
N
6
-phenylisopropyladenosine 0.32>DPCPX 3.2=xanthine amine congener 2.4=
N
6
-cyclopentyladenosine 2.67>5′-(
N
-ethylcarboxamido)-adenosine 7.35>>2-[
p
-(2-carboxyethyl)-phenethyl-amino]-5′-(
N
-ethylcarboxamido)-adenosine 1000>theophylline 83 000. This order of potency fits the criteria for the A
1
adenosine receptor. S-ENBA, a highly selective A
1
receptor agonist, was used to investigate the effect on isoproterenol-mediated vasorelaxation and cAMP accumulation. S-ENBA (0.1 to 10 nmol/L) dose-dependently shifted the isoproterenol-mediated (10
−8
to 10
−5
mol/L) vasorelaxation to the right in vascular rings. S-ENBA (10 nmol/L) inhibited the basal cAMP levels by 36% and attenuated the isoproterenol (10
−5
mol/L)–stimulated cAMP by 25% in the coronary rings. These inhibitory effects of S-ENBA on isoproterenol-mediated cAMP-accumulation and vasorelaxation were abolished by pertussis toxin (100 ng/mL, overnight) treatment of the arteries. Similarly, the effects of S-ENBA on isoproterenol-mediated responses were antagonized by DPCPX. This study provides the evidence suggesting the existence of A
1
adenosine receptors linked to adenylyl cyclase in an inhibitory manner through pertussis toxin–sensitive G proteins in the coronary artery.
Functional Coupling Between Glycolysis and Sarcoplasmic Reticulum Ca
2+
Transport
Authors: Xu Kai Y. , Zweier Jay L. , Becker Lewis C.
Published: June 2012

Abstract
To investigate whether the energy derived from glycolysis is functionally coupled to Ca
2+
active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting
45
Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular
45
Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca
2+
-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated
45
Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca
2+
pump. Addition of a soluble hexokinase ATP trap eliminated
45
Ca transport fueled by exogenous ATP but had markedly less effect on
45
Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more
45
Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca
2+
transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca
2+
homeostasis by driving the SR Ca
2+
pump.
Expression of a Mutation Causing Hypertrophic Cardiomyopathy Disrupts Sarcomere Assembly in Adult Feline Cardiac Myocytes
Authors: Marian A. J. , Yu Q.-T. , Mann D. L. , Graham F. L. , Roberts R.
Published: June 2012

Abstract
Mutations in the β-myosin heavy chain (βMyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant βMyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length βMyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg
403
Gln) was induced in the βMyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant βMyHC cDNAs were cloned into pΔE1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/βMyHC-N and Ad5/CMV/βMyHC-403) were generated through homologous recombination of pΔE1spIB/CMV/βMyHC-N or Ad5/CMV/βMyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the βMyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was >95%. Expression of the βMyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription–PCR. The net total protein and β-myosin synthesis were determined by using the amount of incorporation of [
3
H]phenylalanine into total protein and β-myosin, respectively. Although the total amount of protein synthesis was equal among experimental groups, the net myosin synthesis at 48 hours was greater in cardiac myocytes infected with normal or mutant βMyHC constructs than control myocytes or those infected with vector alone (
P
<.05). Electron microscopic examination showed only minor changes in the structure of sarcomeres in all experimental groups at 48 hours after infection. However, disruption of the sarcomeric structures at 120 hours after infection with the mutant βMyHC construct was observed in ≈50% of the myocytes examined, whereas the structure of the sarcomeres remained largely intact in myocytes infected with normal βMyHC construct, adenoviral vector alone, or control cardiocytes. Similar results were confirmed by immunofluorescence using MF-20 antibody to myosin. The results of this study indicate that disruption of sarcomere assembly and myofibrillar organization due to mutant βMyHC protein is the primary defect in HCM.
Requirement for Protein Kinase C Activation in Basic Fibroblast Growth Factor–Induced Human Endothelial Cell Proliferation
Authors: Kent K. Craig , Mii Shinsuke , Harrington Elizabeth O. , Chang James D. , Mallette Sheila , Ware J. Anthony
Published: June 2012

Abstract
The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)–induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca
2+
increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca
2+
and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca
2+
, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca
2+
). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reverse-transcribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-δ, -η, -θ, and -ζ) as well as small amounts of the conventional (Ca
2+
-regulated) isoenzymes PKC-α and -β. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca
2+
, suggests that one or more of these Ca
2+
-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.
Some Growth Factors Stimulate Cultured Adult Rabbit Ventricular Myocyte Hypertrophy in the Absence of Mechanical Loading
Authors: Decker Robert S. , Cook Melissa G. , Behnke-Barclay Monica , Decker Marlene L.
Published: June 2012

Abstract
Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-β1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with β-MHC synthesis being elevated and α-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to “conditioning” by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.