Verapamil potentiates vagally mediated sinoatrial chronotropic responses in dogs

A brief burst of electrical stimuli delivered to the vagus nerve during the cardiac cycle elicits a triphasic cardiac chronotropic response. The cardiac cycle length initially increases, then briefly decreases, and subsequently increases again. We studied the effects of a calcium channel blocking agent, verapamil, on these responses to vagal stimulation during sinoatrial nodal rhythm in anesthetized, open-chest dogs. Verapamil increased the basal cardiac cycle length only slightly; however, the primary cardioinhibition was accentuated approximately 40% (from 396 to 555 ms) by verapamil. Neither the acceleratory phase of this triphasic response nor the secondary cardioinhibition was significantly affected by verapamil. These results indicate that verapamil potentiates the initial action of acetylcholine at the sinoatrial node when the vagus is activated with brief stimuli.     

Time delay of vagally mediated cardiac baroreflex response varies with autonomic cardiovascular control.

To examine whether changes in autonomic activity have an effect on the latency of the vagally mediated cardiac baroreflex response in humans, we investigated the effects of neck suction fluctuating sinusoidally at 0.2 Hz on R-R intervals (known to be mediated mainly by vagal activity) in the supine position, during 15 degrees head-down tilt and 60 degrees head-up tilt, and during vagotonic (2 microg/kg) and vagolytic (10 microg/kg) doses of atropine while the subjects breathed at 0.25 Hz. The phase shift between fluctuations in neck chamber pressure and in R-R interval was calculated by complex transfer function analysis and was used as a measure of the time delay between carotid baroreceptor stimulation and cardiac effector response. Cardiac baroreflex responsiveness increased significantly during low-dose atropine and decreased during head-up tilt or 10 microg/kg atropine. With increasing tilt angle, the time delay between cyclic baroreceptor stimulation and oscillations in R-R interval increased from 0.32 +/- 0.27 s (head down), to 0.59 +/- 0.25 s (supine position, P < 0.05 vs. head down), and to 0.86 +/- 0.27 s (head up, P < 0.01 vs. supine). Low-dose atropine had a similar effect to head-down tilt on baroreflex latency, whereas 10 microg/kg atropine increased the time delay markedly to 1.24 +/- 0.30 s. Our results demonstrate that changes in autonomic activity, generated either by gravitational stimulus or by atropine, not only affect baroreflex responsiveness but also have a major influence on the latency of the vagally mediated carotid baroreceptor-heart rate reflex. The prolonged baroreflex latency during decreased parasympathetic function may contribute to an unstable regulation of heart rate in patients with cardiac disease.     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

A vagally mediated response to metabolic acidosis in anesthetized rabbits

Pentobarbital sodium-anesthetized rabbits received 10-min infusions of acetic, lactic, or propionic acid delivered via a catheter to the right atrium at a rate of 1 mmol/min (n = 14). Arterial [H+] increased by 35.8 +/- 7.6 (SD) nmol/l, a decrease in pH of 0.27 +/- 0.04. By the end of the infusion period respiratory frequency (f), tidal volume (VT), and minute ventilation (V) had increased by 15.5 +/- 6.2 breaths/min, 7.3 +/- 2.7 ml, and 0.86 +/- 0.34 l/min, respectively. Arterial PCO2 (PaCO2) increased initially, but isocapnia was established during the latter half of the infusion (delta PaCO2 = 0.4 +/- 2.0 Torr). Bilateral cervical vagotomy eliminated the f response to acid infusions (n = 9, delta f = 0.6 +/- 2.4 breaths/min). The increase in VT (12.6 +/- 3.1 ml) was greater, but that in V (0.39 +/- 0.11 l/min) was less than in intact animals (P less than 0.05). PaCO2 remained elevated throughout the infusion (delta PaCO2 = 5.5 +/- 2.6 Torr), resulting in a greater rise in arterial [H+] (delta[H+]a = 53.6 +/- 6.6 nmol/l, delta pHa = -0.37 +/- 0.04). It is concluded that vagal afferents play a role in the f response to acute metabolic acidosis in rabbits.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.     

Insulin modulation of chronotropic response to Adrenaline in diabetic dogs.

In 8 unanesthetized dogs, 10-21 days post pancreatectomy, the cardiac chronotropic response to rapid infusion of a pharmacological dosage of Adrenaline was begun. During the subsequent month, the response was recorded electrocardiographically on 19 occasions. On 8 occasions, animals were treated with continuous intravenous administration of fluids and insulin up to the time of the test; on 11, insulin was omitted for 18 hours before Adrenaline injection. Insulin treated animals responded with the typical brief initial bradycardia, followed by some 2 minutes of ventricular tachycardia, and restoration of preinjection heart rate and electrocardiograph pattern within 5 minutes. On those occasions when insulin was omitted, the tachycardia was replaced by ventricular bradycardia. The altered chronotropic response of non-insulin treated dogs indicates impairment of their cardiac beta receptors.     

Use of calcium channel antagonists as magnetoprotective agents

Human polymorphonuclear leukocytes (PMNs) exhibited a time-dependent (0 to 60 min) increase in the release of lysozyme and lactate dehydrogenase (degranulation) when exposed to a static (direct current) magnetic field of 0.1 Tesla. When 1 X 10(6) PMNs were treated with the calcium channel antagonists diltiazem, nifedipine, and verapamil before exposure to a magnetic field, no significant change in degranulation was detected compared to control and sham-exposed PMNs that were similarly treated. Likewise, magnetic field-induced inhibition of cell migration was prevented with the addition of these antagonists. Such changes in degranulation and cell migration occurred in a dose-dependent manner. These results indicated that these agents protected PMNs exposed to a magnetic field, and the damage to the cells that is mediated by magnetic field-stimulated Ca2+ influx might be preventable. In this regard, pharmaceutical agents might prove useful in protection against injurious electromagnetic field exposure.     

The effect of epidermal growth factor on chronotropic response in cardiac cells in culture

Cardiac chronotropic response to epidermal growth factor (EGF) was assessed in chick embryonic ventricular cell aggregates. EGF at a concentration of 10 ug/mL but not at 5 ug/mL produced a significant (p less than 0.05) increase in cardiac beating rate. This was evident within 10 min, reached a peak at about 15 min and remained at that level for 1.5 hr or the rest of the observation period. The effect of EGF on cardiac automaticity was reduced but not abolished at a lower temperature (22oC) that is known to decrease the affinity of the EGF receptor and reduce the internalization of EGF. Hypothermia did not change the maximum increase in heart rate response from isoproterenol although it altered the pattern of the response. Beta adrenoreceptor blockade with metoprolol only slightly altered the response to EGF. These data indicate that EGF produces functional effects on the heart that may be mediated through EGF receptor linked mechanisms.     

Involvement of the fastigial nuclei in vagally mediated respiratory responses

Xu, Fadi, and Donald T. Frazier. Involvement of thefastigial nuclei in vagally mediated respiratory responses.J. Appl. Physiol. 82(6):1853-1861, 1997.Previous studies have demonstrated that thecerebellum, especially the fastigial nucleus (FN), is capable ofmodulating respiratory responses to chemical and mechanical stimuli.Because there is evidence to show projections from vagal afferents tothe FN, the goal of this study was to determine the role of the FN inthe respiratory reflexes elicited by activation of vagal afferents.Experiments were performed in anesthetized (chloralose), paralyzed, andartificially ventilated cats with an occipital exposure of thecerebellum. Administration of capsaicin (Cap; 5-10 µg/kg) viathe right external jugular vein at the end of inspiration andapplication of lung inflation (LI; 10 cmH₂O) during inspiration werecarried out to stimulate nonmyelinated and myelinated vagal afferents,respectively. The phrenic neurogram was recorded as anindex of the respiratory motor output. Control cardiorespiratoryvariables [expiratory duration(TE), arterial bloodpressure] and their immediate responses to stimuli were comparedbefore and after bilateral lesions of the FN. The results showed thefollowing. 1) Capinjection and LI resulted in a dramatic increase inTE (apnea).2) FN lesions did not significantlyalter the control TE; however,the apneic duration induced by Cap injection was prolonged.3) Neither FN lesions norcerebellectomy affected the apneic duration that resulted fromapplication of LI. 4) Cold blockadeof the vagi (6-8°C) eliminated the respiratory responses elicited by LI but not Cap injection; vagotomy abolished the responses to both stimuli. 5) FN lesions didnot change the control ABP or its responses to either LI or Capinjection. It is concluded that the FN is involved in vagally mediatedrespiratory reflexes elicited by activation of nonmyelinated (C-fiber)vagal afferents.

     

Depression of posttetanic twitch potentiation by low calcium and calcium channel antagonists

The purpose of this investigation was to determine if antagonizing extracellular calcium influx altered posttetanic twitch potentiation (PTP). Whole muscles and muscle fiber bundles (less than or equal to 25 fibers) dissected from frog sartorius and semitendinosus muscles were mounted at optimal length in a normal Ringer solution (NR). To determine PTP, isometric twitches were evoked every 10 s (0.1 Hz) before and after a 2.5-s tetanic contraction (80 Hz). To antagonize calcium influx, low-calcium Ringer [LCR, calcium replaced by 3 mM magnesium and 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], NR plus diltiazem (Dilt, 30 microM), NR plus nifedipine (Nif, 10 microM), and NR plus D 600 (30 microM) were also used (n = 8 for each condition). These conditions altered pretetanic twitch tension by only -1.2 +/- 2.4, 4.2 +/- 2.3, 4.7 +/- 3.7, and 1.6 +/- 3.7% (SE), (LCR, Dilt, Nif, and D 600, P greater than 0.05) but caused a noticeable decrease in tension at the end of the tetanus. Under NR conditions, twitches evoked immediately after the tetanus were potentiated by 49.5 +/- 0.4% with the peak rate of tension development (dP/dt) increased by 44.9 +/- 0.5% (P less than 0.05). Antagonizing calcium influx depressed the PTP response by 59.8 +/- 6.2, 55.9 +/- 10.1, 73.2 +/- 6.8, and 29.8 +/- 3.6% (P less than 0.05) and increased dP/dt by 65.8 +/- 11.1, 45.7 +/- 8.6, 55.6 +/- 4.4% and 49.0 +/- 10.5% (P less than 0.05). Addition of drugs immediately after the tetanus only slightly reduced PTP but accelerated recovery of the twitch.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional interactions between organic calcium channel antagonists in smooth muscle

The Ca2+ channel antagonists D-600, diltiazem, and nifedipine are competitive antagonists of Ca2+ responses in K+-depolarized guinea pig taenia coli and rat mesenteric artery preparations. pA2 values for D-600, diltiazem, and nifedipine in taenia coli were 8.28, 7.44, and 9.27, respectively and in mesenteric artery, 9.6, 7.83, and 10.4, respectively. The combination of nifedipine plus diltiazem gave in both tissues antagonism greater than that calculated on the basis of additivity. This suggests, consistent with published 3H-labelled radioligand binding data, that diltiazem and nifedipine interact at distinct sites. However, the combination nifedipine plus D-600 yielded antagonism consistent with additivity of response.     

Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells

Inhibition of whole-cell calcium currents in enzymatically dispersed frog atrial myocytes by D-600, diltiazem, and nifedipine was studied using a single-micropipette voltage-clamp technique. The objective of these experiments was to test the applicability of a modulated-receptor hypothesis similar to that proposed for local anesthetic interactions with sodium channels to account for the tonic and frequency-dependent interactions of these organic compounds with myocardial calcium channels. Data consistent with such a hypothesis include: (a) prominent use-dependent block of iCa by D-600 and diltiazem, which are predominantly charged at physiological pH; (b) iCa block by an externally applied, permanently charged dihydropyridine derivative is greatly attenuated; (c) all three antagonists produce large negative shifts in the voltage dependence of iCa availability; (d) block of iCa by these compounds is state-dependent; (e) reactivation of iCa in the presence of all three antagonists is biexponential, which suggests that drug-free channels recover with a normal time course and drug-bound channels recover more slowly; and (f) the kinetics of the drug-induced slow iCa recovery process may be determined largely by factors such as size and molecular weight, in addition to lipid solubility of the compounds. Experiments in which the pH was modified, however, reveal some important differences for the interaction of organic calcium antagonists with myocardial calcium channels. Acidification, in addition to changing the proportion of charged and neutral antagonist in solution, was found to selectively antagonize tonic inhibition of iCa by diltiazem and nifedipine, without changing the kinetics of the drug-induced slow iCa reactivation process. It is concluded that two distinct receptor sites may be involved in block of iCa by some of these compounds: a proton-accessible site and a proton-inaccessible site.     

Multiple effects of calcium antagonists on plateau currents in cardiac Purkinje fibers

We studied the influence of Mn, La, and D600 on action potentials and plateau currents in cardiac Purkinje fibers. The Ca antagonists each abolished the second inward current, but they failed to act selectively. Voltage clamp experiments revealed two additional effects: decrease of slow outward current (iotachi) activation, and increase of net outward time-independent plateau current. These effects occurred at inhibitor concentrations used in earlier studies, and were essential to the reconstruction of observed Ca antagonist effects on electrical activity. The inhibitory influence of Mn, La, and D600 on iotachi suggested that iotachi activation might depend upon prior Ca entry. This hypothesis was not supported, however, when [Ca]omicron was varied: elevating [Ca]omicron enhanced Ca entry, but iotachi was nevertheless depressed. Thus, the results suggested instead that Ca antagonists and Ca ions have rather similar effects on iotachi, possibly mediated by changes in membrane surface charge.     

Regulation of the cardiac calcium channel by protein phosphatases

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.     

Effect of ryanodine on cardiac calcium current and calcium channel gating current.

The effects of 100 microM ryanodine on the L-type calcium channel were studied using the pacth-clamp technique in isolated guinea pig ventricular myocytes. The inactivation kinetics of the calcium current were slowed down in the presence of ryanodine in agreement with the blockade of the release of calcium from the sarcoplasmic reticulum by the drug. The I-V and steady-state inactivation curves of the calcium current were shifted to negative values by ryanodine. A similar shift was observed in the activation and inactivation curves of the intramembrane charge movement associated with the calcium channel. Due to this shift, ryanodine slightly reduced the maximal amount of displaced charge although it did not modify the transition from the inactivated to the activated state (i.e., charge movement repriming). This result is in notable contrast with that obtained in skeletal muscle, where it has been found that ryanodine interferes with charge movement repriming. These results provide additional evidence of the postulated differences between the architecture of the excitation-contraction coupling system in cardiac and skeletal muscle.     

Effects of hypoxic factors on cardiac chronotropic reactions in the muskrat Ondatra zibethicus in free behavior

Effects of natural and artificial hypoxic factors on cardiac chronotropic reactions were studied in the muskrat Ondatra zibethicus naturally adapted to underwater hypoxia under conditions of free behavior. To record cardiac activity, original implanted ECG sensors designed in the laboratory were used. Under observations were muskrats in the states of rest, movement, swimming on the water surface, diving, underwater swimming, forced underwater immersion, and artificial apnea, in the low-pressure chamber during changes of pressure from 100 to 25 kPa (ascent to an altitude of 11 km) and in the atmosphere of hypoxic mixtures with 5–10% O₂ as well as under conditions of hemic nitrite hypoxia after injection of 3 mg/kg NaNO₂. Heart rate (HR) in muskrats is labile and can change within the limits from 15 to 360 beats/min. A characteristic feature of hypoxic action is development in muskrats of bradycardia that can appear either instantly—both as a conditional reflex and from the nose lobe receptors—or gradually at a decrease of pO₂ in inhaled air. Before diving and after coming to the surface a brief tachycardia can also be observed. The gradual development of tachycardia takes place in nitrite hypoxia. Development of bradycardia was eliminated at blockade of M-cholinoreceptors by atropine, and of tachycardia—at blockade of β-adrenoreceptors by propranolol. Blockade of α-adrenoreceptors by phentolamine did not affect cardiac chronotropic reactions, which indicates the absence of their connection with vasoconstriction. Analysis of the cardiac rhythm variability has revealed a large spectrum of slow cardiointerval fluctuations connected with animal functional states. Regulation of cardiac chronotropic reactions in muskrats under effect of hypoxic factors operates along both sympathetic and parasympathetic pathways of the autonomic nervous system, the leading role in these processes being played by vagus influences. Original Russian Text. V. I. Shereshkov, T. E. Shumilova, D. A. Kuzmin, I. N. Yanvareva, and A. D. Nozdrachev, 2006, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2006, Vol. 42, No. 4, pp. 371–377.     

Mechanism of fluoride action on the L-type calcium channel in cardiac ventricular myocytes

The modulatory actions of fluoride on the function of the dihydropyridine-sensitive (L-type) Ca2+ channel were studied in rabbit cardiac myocytes. In cell-attached voltage-clamp experiments, using barium as the charge carrier, fluoride increased the activity of the Ca2+ channel dose-dependently. Low concentrations (<10 mM) of fluoride increased the number of traces with channel activities, and decreased the number of traces without channel activities, resulting in a net increase in the open-channel probability. The effect of 5 mM fluoride on the Ca2+ channel was inhibited by the presence of non-hydrolyzable guanosine diphosphate analog in the cell. On the other hand, high concentrations (>10 mM) of fluoride increased the open-channel duration, resulting in a marked increase in open-channel probability. A pretreatment of myocytes with a phosphatase inhibitor, okadaic acid, virtually abolished the additional effect of fluoride on the open-channel duration or open probability. A concentration of up to 75 mM fluoride had no effect on the Ca2+-channel activity when the myocytes were pretreated with a potent inhibitor of protein kinases, indicating that fluoride increased the Ca2+- channel activity via modulation of the phosphorylation state of the myocyte or the channel protein alone.