The chronoinotropic effects of new regulatory input to the heart of land pulmonates

The postjunctional potentials and chronoinotropic reactions of the heart evoked by activation of multimodal neurons and/or left pallial nerve were investigated in three species of land pulmonates: Achatina fulica Ferrussac, Helix lucorum L., Arianta arbustorum L. Both spikes of giant homologous neurons (d-VLN, d-RPLN by A. fulica, command neurons of pneumostoma LPa3, RPa3 by H. lucorum) and stimulation of the peripheral end of the left pallial nerve evoked the similar biphasic inhibitory-excitatory junction potentials in the heart, in mantle muscles and in different parts of visceral complex. The positive chronoinotropic effects of this input in the hearts of whole-mount preparations were modified due to interaction with well-known neural cardioregulating network of the system of intestinal nerve.     

Neuroeffector connections of multimodal neurons in the African snail (Achatina fulica)

Using a new method of animal preparation, the efferent connections of giant paired neurons on the dorsal surface of visceral and right parietal ganglia of snail, Achatina fulica, were examined. It was found that spikes in giant neurons d-VLN and d-RPLN evoke postjunctional potentials in different points of the snail body and viscerae (in the heart, in pericardium, in lung cavity and kidney walls, in mantle and body wall muscles, in tentacle retractors and in cephalic artery). The preliminary analysis of synaptic latency and facilitation suggests a direct connections between giant neurons and investigated efferents.     

Axonal pathways of giant neurons identified in the right parietal and visceral ganglia in the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac)

• 1.1. The axonal pathways of thirteen giant neurons identified in the right parietal and the visceral ganglia, found in the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac), were investigated by intracellular injections of Lucifer Yellow, with regard to their axonal projections into the following six peripheral nerves: lap n (left anterior palliai nerve), lpp n (left posterior palliai nerve), int n (intestinal nerve), anal n (anal nerve), rpp n (right posterior palliai nerve) and rap n (right anterior palliai nerve).
• 2.2. These projections were confirmed by the recording of the axonal responses from the nerves.
• 3.3. On the dorsal surface of the right parietal ganglion, the following four giant neurons were identified: PON (periodically oscillating neuron), TAN (tonically autoactive neuron), RAPN (right anterior palliai neuron), and d-RPLN (dorsal-right parietal large neuron).
• 4.4. The PON axonal pathways projected into int n; those of TAN into all of the nerves examined; those of RAPN into lap n, lpp n, int n, anal n and rap n.; and those of d-RPLN into pd nn (pedal nerves) through the pedal ganglia, lpp n, anal n, rap n and sometimes lap n.
• 5.5. On the dorsal surface of the visceral ganglion, the following four giant neurons were also identified: VIN (visceral intermittently firing neuron), FAN (frequently autoactive neuron), INN (intestinal nerve neuron) and d-VLN (dorsal-visceral large neuron).
• 6.6. The VIN axonal pathways, which had no branch into the six nerves examined, went to both the right and the left pedal ganglia, sending a branch into the cerebro-pleural connective; those of FAN projected into lap n, anal n and rap n, and sometimes into lpp n and rpp n; those of INN into int n; and those of d-VLN into pd nn, lap n, lpp n, anal n and rap n.
• 7.7. On the ventral surface of the right parietal ganglion, v-RPLN (ventral-right parietal large neuron) was identified. The axonal pathways went to pd nn, lap n, lpp n, anal n and rap n.
• 8.8. On the ventral surface of the visceral ganglion, the four giant neurons, v-VNAN (ventral-visceral noisy autoactive neuron), v-VLN (ventral-visceral large neuron), r-VMN (right-visceral multiple spike neuron) and 1-VMN (left-visceral multiple spike neuron) were identified.
• 9.9. The axonal pathway of v-VNAN projected into rpp n and rap n; those of v-VLN into pd nn, lap n, anal n, rap n and sometimes to lpp n; those of r-VMN into int n and rpp n; and those of 1-VMN also into int n and rpp n.
• 10.10. The present morphologial investigations of the giant neurons confirmed well the identifications of the neurons previously studied. The axon of the neurons examined here, except for VIN, projected into some of the peripheral nerves, while the VIN axon extended into the cerebro-pleural connective.
• 11.11. The five neurons, PON, TAN, v-VNAN, r-VMN and 1-VMN, formed fine axonal arborizations terminating at the neuropile, while the arborizations of the other neurons were not clearly observed.
• 12.12. Although the anatomical structures of the portion examined of the suboesophageal ganglia are asymmetrical, three pairs of symmetrically-situated neurons, d-RPLN and d-VLN, v-RPLN and v-VLN, and r-VMN and 1-VMN, were found, indicating the existence of symmetrical components in the ganglia.

     

Further study of effects of synthetic peptides on identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

1. Effects of the following peptides at 10(-4) M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14-27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, gamma-melanocyte stimulating hormone. 2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone). 3. Of the peptides examined, only Arg-vasotocin at 10(-4) M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone. 4. The other peptides examined were completely ineffective on all of the neurones tested.     

Brain stem mechanisms underlying acupuncture modality-related modulation of cardiovascular responses in rats.

The present study was designed to investigate brain stem responses to manual acupuncture (MA) and electroacupuncture (EA) at different frequencies at pericardial P (5-6) acupoints located over the median nerve. Activity of premotor sympathetic cardiovascular neurons in the rostral ventral lateral medulla (rVLM) was recorded during stimulation of visceral and somatic afferents in ventilated anesthetized rats. We stimulated either the splanchnic nerve at 2 Hz (0.1-0.4 mA, 0.5 ms) or the median nerve for 30 s at 2, 10, 20, 40, or 100 Hz using EA (0.3-0.5 mA, 0.5 ms) or at approximately 2 Hz with MA. Twelve of 18 cells responsive to splanchnic and median nerve stimulation could be antidromically driven from the intermediolateral columns of the thoracic spinal cord, T2-T4, indicating that they were premotor sympathetic neurons. All 18 neurons received baroreceptor input, providing evidence of their cardiovascular sympathoexcitatory function. Evoked responses during stimulation of the splanchnic nerve were inhibited by 49 +/- 6% (n = 7) with EA and by 46 +/- 4% (n = 6) with MA, indicating that the extent of inhibitory effects of the two modalities were similar. Inhibition lasted for 20 min after termination of EA or MA. Cardiovascular premotor rVLM neurons responded to 2-Hz electrical stimulation at P 5-6 and to a lesser extent to 10-, 20-, 40-, and 100-Hz stimulation (53 +/- 10, 16 +/- 2, 8 +/- 2, 2 +/- 1, and 0 +/- 0 impulses/30 stimulations, n = 7). These results indicate that rVLM premotor sympathetic cardiovascular neurons that receive convergent input from the splanchnic and median nerves during low-frequency EA and MA are inhibited similarly for prolonged periods by low-frequency MA and EA.     

Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium

A strong premature electrical stimulus (S(2)) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca(2+) concentration ([Ca(2+)](i)) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca(2+)](i) optical mapping and for vulnerable window determination. After baseline pacing (S(1)), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S(2) was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca(2+)](i) varied depending on the S(2) polarity. Relative to the level of [Ca(2+)](i) during the S(1) beat, the [Ca(2+)](i) level 40 ms after the onset of monophasic S(2) increased by 36+/-8% at virtual anodes and 20+/-5% at virtual cathodes (P<0.01), compared with 25+/-5% at both virtual cathode-anode and anode-cathode sites for biphasic S(2). The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S(2) than for either anodal or cathodal S(2) (n=7, P<0.01). Treatment with thapsigargin and ryanodine (n=6) significantly prolonged the action potential duration compared with control (255+/-22 vs. 189+/-6 ms, P<0.05) and eliminated the difference in VFT between monophasic and biphasic S(2), although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca(2+)](i) than virtual cathodes. Monophasic S(2) is associated with lower VFT than biphasic S(2), but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca(2+)](i) dynamics modulate, but are not essential, to ventricular vulnerability.     

Weight loss increases cardiovagal baroreflex function in obese young and older men

We tested the hypothesis that reductions in total body and abdominal visceral fat with energy restriction would be associated with increases in cardiovagal baroreflex sensitivity (BRS) in overweight/obese older men. To address this, overweight/obese (25 < or = body mass index < or = 35 kg/m(2)) young (OB-Y, n = 10, age = 32.9 +/- 2.3 yr) and older (OB-O, n = 6, age = 60 +/- 2.7 yr) men underwent 3 mo of energy restriction at a level designed to reduce body weight by 5-10%. Cardiovagal BRS (modified Oxford technique), body composition (dual-energy X-ray absorptiometry), and abdominal fat distribution (computed tomography) were measured in the overweight/obese men before weight loss and after 4 wk of weight stability at their reduced weight and compared with a group of nonobese young men (NO-Y, n = 13, age = 21.1 +/- 1.0 yr). Before weight loss, cardiovagal BRS was approximately 35% and approximately 60% lower (P < 0.05) in the OB-Y and OB-O compared with NO-Y. Body weight (-7.8 +/- 1.1 vs. -7.3 +/- 0.7 kg), total fat mass (-4.1 +/- 1.0 vs. -4.4 +/- 0.8 kg), and abdominal visceral fat (-27.6 +/- 6.9 vs. -43.5 +/- 10.1 cm(2)) were reduced (all P < 0.05) after weight loss, but the magnitude of reduction did not differ (all P > 0.05) between OB-Y and OB-O, respectively. Cardiovagal BRS increased (11.5 +/- 1.9 vs. 18.5 +/- 2.6 ms/mmHg and 6.7 +/- 1.2 vs. 12.8 +/- 4.2 ms/mmHg) after weight loss (both P < 0.05) in OB-Y and OB-O, respectively. After weight loss, cardiovagal BRS in the obese/overweight young and older men was approximately 105% and approximately 73% (P > 0.05) of NO-Y (17.5 +/- 2.2 ms/mmHg). Therefore, the results of this study indicate that weight loss increases the sensitivity of the cardiovagal baroreflex in overweight/obese young and older men.     

乙酰胆碱对蟾蜍背根神经节神经元膜电位的影响及离子机制

在离体灌流的蟾蜍背根神经节(DRG)标本上,用微电极进行胞内记录。在73个神经元中,依神经纤维的传导速度将神经元分为 A 型及 C 型,其中 A 型细胞67个,C 型6个,静息膜电位为-67.5±1.3mV((?)±SE)。当加4×10~(-4)—6×10~(-4)mol/L 乙酰胆碱(ACh),可观察到如下四种膜电位变化:1.超极化:幅值9.1±3.0mV((?)±SE,n=23);(2)去极化:幅值12.9±2.2mV((?)+SE,n=20);(3)双相反应(n=24):先超极化,后去极化,超极化幅值8.0±2.4mV((?)+SE),去极化幅值10.9±3.1mV((?)±SE);(4)无反应(n=6)。用阿托品(1.3×10~(-5)mol/L,n=23),或同时应用筒箭毒与六甲双铵(浓度均为1.4×10~(-5)mol/L,n=8)灌流,能分别阻断 ACh 引起的膜的超极化或去极化。ACh 引起超极化反应时膜电导平均增加13.8%,翻转电位值大约-96mV。四乙铵(TEA,20mmol/L)能使 ACh 的去极化幅值增加48.2±3.2%((?)±SE,n=6),超极化幅值减小79.4±4.3%((?)±SE,n=8)。MnCl_2(4mmol/L)使 ACh 的去极化及超极化幅值分别减小54.2±7.2%((?)±SE,n=5)及69.2±6.4%((?)±SE,n=14)。以上结果提示:ACh 引起的 DRG 神经细胞膜去极化反应由 N 型乙酰胆碱受体介导,而超极化反应由 Μ 型乙酰胆碱受体介导,前者可能包含了多种离子电导的改变,后者则可能与钾电导增加有关。     

Effects of mechanical uncouplers, diacetyl monoxime, and cytochalasin-D on the electrophysiology of perfused mouse hearts

Chemical uncouplers diacetyl monoxime (DAM) and cytochalasin D (cyto-D) are used to abolish cardiac contractions in optical studies, yet alter intracellular Ca(2+) concentration ([Ca(2+)](i)) handling and vulnerability to arrhythmias in a species-dependent manner. The effects of uncouplers were investigated in perfused mouse hearts labeled with rhod-2/AM or 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) to map [Ca(2+)](i) transients (emission wavelength = 585 +/- 20 nm) and action potentials (APs) (emission wavelength > 610 nm; excitation wavelength = 530 +/- 20 nm). Confocal images showed that rhod-2 is primarily in the cytosol. DAM (15 mM) and cyto-D (5 microM) increased AP durations (APD(75) = 20.0 +/- 3 to 46.6 +/- 5 ms and 39.9 +/- 8 ms, respectively, n = 4) and refractory periods (45.14 +/- 12.1 to 82.5 +/- 3.5 ms and 78 +/- 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min and DAM by 10% gradually in 1 h (n = 5 each). Uncouplers did not alter the direction and gradient of repolarization, which progressed from apex to base in 15 +/- 3 ms. Peak systolic [Ca(2+)](i) increased with cyto-D from 743 +/- 47 (n = 8) to 944 +/- 17 nM (n = 3, P = 0.01) but decreased with DAM to 398 +/- 44 nM (n = 3, P < 0.01). Diastolic [Ca(2+)](i) was higher with cyto-D (544 +/- 80 nM, n = 3) and lower with DAM (224 +/- 31, n = 3) compared with controls (257 +/- 30 nM, n = 3). DAM prolonged [Ca(2+)](i) transients at 75% recovery (54.3 +/- 5 to 83.6 +/- 1.9 ms), whereas cyto-D had no effect (58.6 +/- 1.2 ms; n = 3). Burst pacing routinely elicited long-lasting ventricular tachycardia but not fibrillation. Uncouplers flattened the slope of AP restitution kinetic curves and blocked ventricular tachycardia induced by burst pacing.     

The Gurvich waveform has lower defibrillation threshold than the rectilinear waveform and the truncated exponential waveform in the rabbit heart

Implantable cardioverter defibrillator studies have established the superiority of biphasic waveforms over monophasic waveforms. However, external defibrillator studies of biphasic waveforms are not as widespread. Our objective was to compare the defibrillation efficacy of clinically used biphasic waveforms, i.e., truncated exponential, rectilinear, and quasi-sinusoidal (Gurvich) waveforms in a fibrillating heart model. Langendorff-perfused rabbit hearts (n = 10) were stained with a voltage-sensitive fluorescent dye, Di-4-ANEPPS. Transmembrane action potentials were optically mapped from the anterior epicardium. We found that the Gurvich waveform was significantly superior (p < 0.05) to the rectilinear and truncated exponential waveforms. The defibrillation thresholds (mean +/- SE) were as follows: Gurvich, 0.25 +/- 0.01 J; rectilinear-1, 0.34 +/- 0.01 J; rectilinear-2, 0.33 +/- 0.01 J; and truncated exponential, 0.32 +/- 0.02 J. Using optically recorded transmembrane responses, we determined the shock-response transfer function, which allowed us to predict the cellular response to waveforms at high accuracy. The passive parallel resistor-capacitor model (RC-model) predicted polarization superiority of the Gurvich waveform in the myocardium with a membrane time constant (taum) of less than 2 ms. The finding of a lower defibrillation threshold with the Gurvich waveform in an in vitro model of external defibrillation suggests that the Gurvich waveform may be important for future external defibrillator designs.     

Cardiac vagal modulation of heart rate during prolonged submaximal exercise in animals with healed myocardial infarctions: effects of training

The present study investigated the effects of long-duration exercise on heart rate variability [as a marker of cardiac vagal tone (VT)]. Heart rate variability (time series analysis) was measured in mongrel dogs (n = 24) with healed myocardial infarctions during 1 h of submaximal exercise (treadmill running at 6.4 km/h at 10% grade). Long-duration exercise provoked a significant (ANOVA, all P < 0.01, means +/- SD) increase in heart rate (1st min, 165.3 +/- 15.6 vs. last min, 197.5 +/- 21.5 beats/min) and significant reductions in high frequency (0.24 to 1.04 Hz) power (VT: 1st min, 3.7 +/- 1.5 vs. last min, 1.0 +/- 0.9 ln ms(2)), R-R interval range (1st min, 107.9 +/- 38.3 vs. last min, 28.8 +/- 13.2 ms), and R-R interval SD (1st min, 24.3 +/- 7.7 vs. last min 6.3 +/- 1.7 ms). Because endurance exercise training can increase cardiac vagal regulation, the studies were repeated after either a 10-wk exercise training (n = 9) or a 10-wk sedentary period (n = 7). After training was completed, long-duration exercise elicited smaller increases in heart rate (pretraining: 1st min, 156.0 +/- 13.8 vs. last min, 189.6 +/- 21.9 beats/min; and posttraining: 1st min, 149.8 +/- 14.6 vs. last min, 172.7 +/- 8.8 beats/min) and smaller reductions in heart rate variability (e.g., VT, pretraining: 1st min, 4.2 +/- 1.7 vs. last min, 0.9 +/- 1.1 ln ms(2); and posttraining: 1st min, 4.8 +/- 1.1 vs. last min, 2.0 +/- 0.6 ln ms(2)). The response to long-duration exercise did not change in the sedentary animals. Thus the heart rate increase that accompanies long-duration exercise results, at least in part, from reductions in cardiac vagal regulation. Furthermore, exercise training attenuated these exercise-induced reductions in heart rate variability, suggesting maintenance of a higher cardiac vagal activity during exercise in the trained state.     

The effect of lidocaine compared with verapamil on the enhanced ventricular rhythm in the infarcted canine heart

Myocardial ischemia was produced in dogs by the occlusion of the left anterior descending (LAD) coronary artery for 24 or 48 h. After complete atrioventricular block was produced, enhanced ventricular rhythm was observed in all animals. The enhanced ventricular rhythm showed multiple QRS configurations and had spontaneous cycle lengths (SCL) of 397 +/- 18 ms (n = 20) after 24 h of LAD occlusion and 446 +/- 23 ms (n = 20) after 48 h of LAD occlusion. Overdrive pacing did not result in the termination of the enhanced ventricular rhythm in any experiment. Propranolol, as a cumulative dose of 1.5-2.0 mg/kg i.v., also did not abolish the enhanced ventricular rhythm. In 24-h infarcted hearts, lidocaine abolished the enhanced ventricular rhythm in 1 of 11 experiments. In the remaining 10 experiments, the ventricular SCL was increased from 401 +/- 22 to 491 +/- 26 ms after a cumulative dose of 8.8 +/- 0.7 mg/kg of lidocaine. In the presence of verapamil, given as a cumulative dose of 0.60 +/- 0.11 mg/kg, the ventricular SCL was increased from 401 +/- 33 to 482 +/- 64 ms (n = 9). In 48-h infarcted hearts, lidocaine abolished the enhanced ventricular rhythm in 5 of 11 experiments. Both lidocaine and verapamil increased the SCL of hearts in which the enhanced ventricular rhythm persisted. Analysis of variance showed that only the increase in SCL by lidocaine in 48-h infarcted hearts was statistically significant. The atrial and idioventricular rhythms in noninfarcted hearts responded differently to lidocaine and verapamil. The results suggest that some electrophysiological effects of antiarrhythmic drugs in the normal heart may not be applicable to those in the diseased situation.     

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.     

The role of inhibitory heterotrimeric G proteins in the control of in vivo heart rate dynamics

Multiple isoforms of inhibitory Galpha-subunits (Galphai1,2,3, as well as Galphao) are present within the heart, and their role in modulating pacemaker function remains unresolved. Do inhibitory Galpha-subunits selectively modulate parasympathetic heart rate responses? Published findings using a variety of experimental approaches have implicated roles for Galphai2, Galphai3, and Galphao in parasympathetic signal transduction. We have compared in vivo different groups of mice with global genetic deletion of Gialpha1/Galphai3, Galphai2, and Galphao against littermate controls using implanted ECG telemetry. Significant resting tachycardia was observed in Galphai2(-/-) and Galphao(-/-) mice compared with control and Galphai1(-/-)/Galphai3(-/-) mice (P < 0.05). Loss of diurnal heart rate variation was seen exclusively in Galphao(-/-) mice. Using heart rate variability (HRV) analysis, compared with littermate controls (4.02 ms2 +/- 1.17; n = 6, Galphai2(-/-)) mice have a selective attenuation of high-frequency (HF) power (0.73 ms2 +/- 0.31; n = 5, P < 0.05). Galphai1(-/-)/Galphai3(-/-) and Galphao(-/-) cohorts have nonsignificant changes in HF power. Galphao(-/-) mice have a different basal HRV signature. The observed HRV phenotype in Galphai2(-/-) mice was qualitatively similar to atropine (1 mg/kg)-treated controls [and mice treated with the GIRK channel blocker tertiapinQ (0.05 mg/kg)]. Maximal cardioinhibitory response to the M(2)-receptor agonist carbachol (0.5 mg/kg) compared with basal heart rate was attenuated in Galphai2(-/-) mice (0.08 +/- 0.04; n = 6) compared to control (0.27 +/- 0.04; n = 7 P < 0.05). Our data suggest a selective defect of parasympathetic heart rate modulation in mice with Galphai2 deletion. Mice with Galphao deletion also have a defect in short-term heart rate dynamics, but this is qualitatively different to the effects of atropine, tertiapinQ, and Galphai2 deletion. In contrast, Galphai1 and Galphai3 do not appear to be essential for parasympathetic responses in vivo.     

Role of cAMP in activation of ischemically sensitive abdominal visceral afferents

A number of metabolites produced during abdominal ischemia can stimulate and/or sensitize visceral afferents. The precise mechanisms whereby these metabolites act are uncertain. Other studies have shown that the adenylate cyclase-cAMP system may be involved in the activation of sensory neurons. Therefore, we hypothesized that cAMP contributes to the activation of ischemically sensitive abdominal visceral afferents. Single-unit activity of abdominal visceral C fibers was recorded from the right thoracic sympathetic chain in anesthetized cats before and during 7 min of abdominal ischemia. Forty-six percent of ischemically sensitive C fibers responded to intra-arterial injection of 8-bromo-cAMP (0.35-1. 0 mg/kg), an analog of cAMP, with responses during ischemia increasing from 0.50 +/- 0.06 to 0.84 +/- 0.08 impulses/s (P < 0.05, n = 11 C fibers). Conversely, an inhibitor of adenylate cyclase, 2', 5'-dideoxyadenosine (DDA; 0.1 mg/kg iv), attenuated ischemia-induced increase in activity of afferents from 0.66 +/- 0.10 to 0.34 +/- 0. 09 impulses/s (P < 0.05; n = 8). Furthermore, whereas exogenous PGE(2) (3-4 microg/kg ia) augmented the ischemia-induced increase in activity of afferents (P < 0.05, n = 10), treatment with DDA (0.1 mg/kg iv) substantially reduced the increase in discharge activity of afferents during ischemia, which was augmented by PGE(2) (1.45 +/- 0.24 vs. 0.70 +/- 0.09 impulses/s, -DDA vs. +DDA; P < 0.05) in six fibers. A time control group (n = 4), however, demonstrated similar increases in the activity of afferents with repeated administration of PGE(2). These data suggest that cAMP contributes to the activation of abdominal visceral afferents during ischemia, particularly to the action of PGs on activation and/or sensitization of these endings.     

Properties of an early outward current in single cells of the mouse ventricle

Single ventricular myocytes of adult mice were prepared by enzymatic dissociation for voltage clamp experiments with the one suction pipette dialysis method. After blocking the Na current by 10(-4) mol/l TTX early outward currents (IEO) with incomplete inactivation could be elicited by clamping from -50 mV to test potentials (VT) positive to -30 mV. Interfering Ca currents were very small (less than 0.6 nA at VT = 0 mV). The approximation of IEO by the q4r-model showed a pronounced decrease in the time constant of activation (tau q) to more positive potentials. At 50 ms test pulses the time course of the incomplete inactivation could be described by two exponentials and a constant. The time constant of the fast exponential (tau r1) showed a slight decline towards more positive test potentials (8.1 +/- 1.0 ms at -10 mV; 5.8 +/- 1.2 ms at +50 mV, mean +/- SD, n = 5) whereas the time constant of the slow exponential (tau r2) was voltage independent (41.1 +/- 7.9 ms, mean +/- SD, n = 5). The contributions of the fast exponential and the pedestal increased towards positive test potentials. The Q10 value for the time constants of activation and fast inactivation was 2.36 +/- 0.19 and 2.51 +/- 0.09 (mean +/- SD, n = 3), respectively. After an initial delay the recovery of IEO at a recovery potential of -50 mV could be fitted monoexponentially with a time constant of 16.3 +/- 2.9 ms (mean +/- SD, n = 3). The time course of the onset of inactivation determined with the double pulse protocol was slower than the decay at the same potential, and could be described as sum of a fast (tau = 18.4 +/- 6.0 ms) and a slow (tau = 62.1 +/- 19.9ms, mean +/- SD, n = 3) exponential. IEO could be blocked completely by 1 mmol/l 4-aminopyridine at potentials up to +20 mV. Stronger depolarizations had an unblocking effect.     

Rate-dependent [K+](o) accumulation in canine right atria in vivo: electrophysiological consequences

Sudden increases in heart rate cause accumulation of K+ in the extracellular space. However, the exact relationship between rate and extracellular K+ concentration ([K+](o)) in vivo is unknown. We measured [K+](o) in right atria of anesthetized dogs by using K(+)-sensitive electrodes. Peak increase in [K+](o) ranged from 0.18 +/- 0.04 mM [means +/- SE; cycle length (CL) = 350 ms] to 0.80 +/- 0.09 mM (CL = 250 ms) above baseline (3.50 +/- 0.08 mM at CL = 380 ms; n = 5). During rapid pacing-induced atrial fibrillation, peak increase in [K+](o) averaged 0.80 +/- 0.07 mM (n = 5). Whole cell current-clamp measurements in single right atrial myocytes (n = 5) showed that raising [K+](o) from 3 to 5 mM in 1-mM steps progressively depolarized resting membrane potential and reduced both phase 0 action potential amplitude and maximal upstroke velocity. Multisite epicardial mapping (n = 4) demonstrated that sudden rate increases changed longitudinal conduction velocity (CV(L)) by -3.6 +/- 1.8% to -5.9 +/- 1.2% over a CL range of 330 to 250 ms. Our observations suggest that rate-related [K+](o) accumulation in vivo is of sufficient magnitude to modulate those cellular electrophysiological properties that determine atrial CV(L).     

Overexpression of SERCA2a accelerates repolarisation in rabbit ventricular myocytes

Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation-contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15-25 M Omega resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481+/-98, n=12; Ad.GFP: 464+/-85, n=11; Ad.SERCA2a: 285+/-69, n=13 (mean+/-S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375+/-64, n=22; Ad.GFP: 363+/-47, n=18; Ad.SERCA2a: 231+/-54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5+/-4.2, n=8; Ad.SERCA2a: 15.5+/-4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (microM/l non-mitochondrial volume): non-infected: 25.5+/-7, n=8; Ad.GFP: 25.7+/-11, n=6; Ad.SERCA2a: 80.5+/-19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.     

Effects of abdominal or cardiopulmonary sympathetic afferents on upper cervical inspiratory neurons

Responses of upper cervical inspiratory neurons (UCINs) to abdominal visceral or cardiopulmonary sympathetic stimulation were studied using extracellular recordings from 213 UCINs in 54 pentobarbital sodium-anesthetized and paralyzed rats. Phrenic nerve activity was used to assess inspiration. The UCINs discharging during inspiration only were mainly in the C(1) segment, whereas phase-spanning UCINs were mostly in the C(2) segment. Phase-spanning activity was typically retained after overventilation or vagotomy. When greater splanchnic nerve (GSN) or cardiopulmonary sympathetic afferent (CPSA) fibers were electrically stimulated, augmented UCIN activity was observed in 65% of cells responding to CPSA stimulation but in only 17% of cells responding to GSN. Response latencies were 10.7 +/- 0.5 and 20.6 +/- 1.5 (SE) ms, respectively. Many augmented responses to CPSA stimulation (64%) and all augmented responses to GSN stimulation were followed by suppression of UCIN discharge (biphasic response). Phrenic nerve activity was suppressed by both GSN and CPSA stimulation, but with shorter latency for the latter (29 +/- 0.7 vs. 14.0 +/- 0.7 ms). Excitation of UCINs using CPSA stimulation occurs more often and by a more direct pathway than for GSN input.     

Ionic current of an identifiable giant neurone, d-RPLN, of an African giant snail (Achatina fulica Férussac), measured under voltage clamping--II. Outward currents

1. The outward currents of d-RPLN (dorsal-right parietal large neurone), one of the largest identifiable neurones of an African giant snail, were studied. 2. At the holding potential (-90 mV) and at the command voltage (Vc20 mV), the current values were 3.05 +/- 0.13 microA (M +/- SE) for the peak (n = 38), and 1.96 +/- 0.10 microA for the plateau (n = 37). 3. The peak time constant (Vc = 0 mV) was 2.05 +/- 0.08 msec. 4. Tetraethylammonium at 50 mM reduced the plateau value up to 50-55% of the normal, but had little effect on the peak. 5. 5-Aminopyridine at 5.0 mM diminished the peak value to about 50-55%, and delayed the peak time. 6. Quinine at 0.25 mM decreased both the peak and the plateau approximately to 55-65% of their controls, but shortened the peak time when Vc was beyond 0 mV, in contrast to the case of 4-AP. 7. The calcium-free state (replaced with cobalt) reduced these currents to about 75% of the normal, and evidently delayed the peak time.