Rabbit Sinus Research Articles

Study of the Effect of Acetylcholine on the Excitability of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Study of the Effect of Acetylcholine on the Excitability of True Pacemaker Cells of Rabbit Sinus Node Using Computer Simulation

Sélection et caractérisation pharmacologique de Procoralan®, un inhibiteur sélectif du courant pacemaker If

The screening of a series of benzocycloalkane derivatives led to the selection of Procoralan® (ivabradine), the first selective inhibitor of the depolarizing If (funny) current of the sinus node, for the treament of myocardial ischaemia. In vitro, this compound reduces the spontaneous beating rate of isolated right rat atria and the firing rate of the action potential of rabbit sinus node preparations. This effect is explained by a reduction in the diastolic depolarisation slope of the action potential and underlies a selective inhibition of the pacemaker If current. In vivo, it induces a selective reduction in heart rate both at rest and during exercise. It preserves myocardial contractility, atrioventricular conduction and ventricular repolarisation duration. Ivabradine exerts a similar anti-ischaemic activity in exercise-induced myocardial ischaemia in pigs to that of a β-blocker and, furthermore, it limits to a greater extent ischaemic myocardial contractile dysfunction.

Study of the Effect of Acetylcholine on Ion Currents in Single Cells of True and Latent Pacemakers of Rabbit Sinus Node using Computer Simulation

Study of the Effect of Acetylcholine on Ion Currents in Single Cells of True and Latent Pacemakers of Rabbit Sinus Node using Computer Simulation

Bone regeneration in rabbit sinus lifting associated with bovine BMP.

Autogenous bone is considered the optimal grafting material for sinus lifting, although its harvesting causes great patient discomfort. Various approaches have been taken in order to obtain sinus lifting with preexisting tissue. However, because of the unsuitability of such tissue, additional materials have been required. Alternatively, biomaterials from humans or other animals are used. In this study, the efficacy of using morphogenetic bovine bone protein (BMPb) to augment the maxillary sinus floor was examined. Four grafting materials were employed: lyophilized bovine bone powder, absorbable collagen flakes, natural hydroxylapatite, and synthetic hydroxylapatite. Two groups of rabbits were studied. In one group, graft material only was used. In the other, graft material was combined with 0.5 mg BMPb. During 8 weeks of observation, polyfluorochrome tracers were injected in subcutaneous tissue to evaluate new bone- deposition periods. Following sacrifice, the samples were examined under fluorescent and light microscopes. Results indicated 33.34% more newly formed bone in BMPb animals than in controls. Graft-material resorption increased, but natural HA showed no significant alterations. The results show that the use of BMPb, although providing osteoinduction, might not promote sufficient bone formation. Nonetheless, this material could provide an alternative to autogenous grafts, thereby avoiding patient discomfort.

Poster 23: Type I collagen, osteocalcin, and osteonectin expression by different bioimplants in a rabbit sinus augmentation model

Poster 23: Type I collagen, osteocalcin, and osteonectin expression by different bioimplants in a rabbit sinus augmentation model

Estimating the active dimension of cardiac pacemaking dynamics

Although there exist several different models for the excitability of cardiac and nerve cells, the number of ionic currents that determine this excitability is not clearly known. On the other hand, the chaotic behavior of excitable cells has recently been demonstrated. The science of complex dynamics provides tools for the reconstruction of the original dynamics from a time series of observed data. We determined the degrees of freedom for the excitability of a single cardiac pacemaker cell by chaotic analysis of a time series of action potentials (APs) recorded from the rabbit sinus node. We provide evidence that at least 12 variables are needed to determine the AP. From our findings, we conclude that a new model for the cardiac pacemaker mechanism may be needed, because previous models could deal only with fewer than 12 variables. Our approach based on our observation of a time series offers a powerful tool for constructing a model for the underlying biological mechanism.

Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node.

This study reports the comparative quantitative, morphological, and electrophysiological properties of two pacemaker cell types, spider and spindle-shaped cells, isolated from the rabbit sinoatrial node. Isolated nodal cells were studied with perforated and ruptured patch whole cell recording techniques. The basic spontaneous cycle length of the spider cells was 381 +/- 12 ms, and the basic spontaneous cycle length of the spindle cells was 456 +/- 17 ms (n = 12, P < 0.05). The spider cells had a more positive maximum diastolic potential (-54 +/- 1 mV) compared with the spindle cells (-68 +/- 1mV, P < 0.05). The overshoot and action potential amplitudes were also smaller in the spider cells. The hyperpolarization-activated inward (I(f)) current density, measured from their tail currents, was 15 +/- 1.3 pA/pF for the spider cells and 9 +/- 0.7 pA/pF for the spindle cells (P < 0.01). I(f) current activation voltage was more positive in the spider cells than the spindle cells. Isoproterenol (1 microM) decreased the spontaneous cycle length of the spider cells by 28 +/- 3% and the spindle cells by 20 +/- 1.5% (P < 0.05). Acetylcholine (0.5 microM) hyperpolarized the membrane potential of the spider cells to -86 +/- 0.7 mV and the spindle cells to -76 +/- 0.8 mV (P < 0.05). In summary, there are at least two distinct pacemaker cell types in the sinus node with different electrophysiological characteristics.

Active ion secretion and permeability of rabbit maxillary sinus epithelium, impact of staphylococcal enterotoxin B

The aim of this study was to investigate the impact of staphylococcal enterotoxin B (SEB), a model superantigen, on the physiologic functions of rabbit maxillary sinus epithelium. Rabbit sinus mucosae were separated under a surgical microscope and mounted in Ussing chambers to record short-circuit current, conductance, and permeability to horseradish peroxidase. The results showed that SEB evoked increases in sinus epithelial cell baseline short-circuit current, conductance, and permeability to horseradish peroxidase. When tumor necrosis factor-α (TNF-α) was added to the Ussing chambers, we got results similar to those obtained by SEB stimulation in vitro; the effects of SEB on sinus epithelial cells could be blocked by pretreatment with anti-TNF-α antibody. These results demonstrate that SEB is able to alter the function of sinus epithelial cells and to affect the capability of the epithelial defensive barrier, which may be mediated by TNF-α. (Otolaryngol Head Neck Surg 2000;123:120-3.)

Mibefradil, an ICa,T blocker, effectively blocks ICa,L in rabbit sinus node cells

To test the hypothesis that the Ca 2+ channel blocker mibefradil slows heart rate due to inhibition of T-type Ca 2+ current in pacemaker cells, we studied effects of mibefradil on action potentials and ionic currents of isolated rabbit sinus node cells using the patch clamp technique. Mibefradil (100 nM and 1 μM) reduced spontaneous rate, decreased action potential amplitude and finally stopped impulse initiation. This action was not due to the drug effect on hyperpolarization-activated pacemaker current, but can be explained by attenuation of both T- and L-type Ca 2+ currents, which were inhibited by mibefradil almost equally (55% and 64% inhibition with 1 μM for T- and L-types, respectively).

Surfactant protein A in rabbit sinus and middle ear mucosa.

In the present study, pulmonary surfactant protein A (SP-A) messenger RNA (mRNA) and protein were characterized in adult rabbit middle ear and maxillary sinus. Fifteen adult rabbits were used for the study: 6 with evidence of acute middle ear infections and maxillary sinusitis, 6 with infections that were successfully treated with tetracycline, and 3 that were pathogen-free. We detected SP-A mRNA in maxillary sinus and middle ear tissues by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR also revealed the presence of SP-B and SP-C mRNA in middle ear and sinus tissues. We detected SP-A protein, of molecular weight approximately 29 and 70 kd, in middle ear and sinus tissues by immunoblot analysis. Unlike the SP-A protein present in the lung, the molecular weight of the SP-A protein present in the middle ear and paranasal sinus was not altered by digestion with an enzyme that cleaves N-linked carbohydrates. Immunostaining and in situ hybridization showed that SP-A protein and mRNA, respectively, were present in surface epithelial cells of the middle ear and in epithelial cells of submucosal glands in sinus tissues. These data provide the first evidence of the presence of pulmonary surfactant proteins in the paranasal sinuses and confirm previous reports of SP-A in the middle ear epithelium.

Microvasculature, blood flow, and vasoreactivity in rabbit sinus mucosa after surgery.

To study the circulatory integrity of the sinus mucosa following surgery, the vascular anatomy, blood flow, and vasoconstrictor response of the regenerated microcirculatory network were analyzed. Forty-six New Zealand White rabbits were operated on unilaterally with either a modified radical operation (MRO) or middle meatal antrostomy (MMA), using the nonoperated sinus for control purposes. After surgery, the animals were left to heal for 1 month. Vascular casts were prepared with a low-viscosity methyl methacrylate resin and studied by scanning electron microscopy. Blood flow was measured by means of radiolabeled microspheres (tin 113 [113Sn]). The vasoconstrictor response to oxymetazoline at increasing concentrations was measured with laser Doppler flowmetry. The number of vessels increased significantly in the regenerated mucosa. The vascular casts displayed a rich microcirculation with local signs of angiogenesis. However, there was no difference in blood flow between the operated cavities and their control sides. Following MRO the regenerated mucosa was more sensitive to the vasoconstrictor effect of oxymetazoline, compared with the control side. This difference was not evident in the MMA-operated sinuses. In this model, surgery does not seriously interfere with the sinus blood flow, although the regenerated mucosa did display an altered vasoreactivity. These findings should be considered in relation to the effects of surgery intended to limit local inflammation and to ensure the ventilation of the sinuses.

Lectin Expression during Wound Healing of the Rabbit Sinus Mucosa: A Study of Regenerating Epithelium and Early Polyp Formation

Lectin expression during wound healing of the rabbit sinus mucosa was examined. Positive UEA-I staining was evident on squamous or cuboidal as well as columnar regenerating epithelial cells (RE cells). PNA staining of columnar RE cells first became evident after neuraminidase treatment, while squamous or cuboidal RE cells stained positively with PNA alone. Fucosylation within RE cells thus occurred from a relatively early period, and sialylation followed at later stages. Ingrowing epithelial cells of early polyp formation stained negatively with UEA-I, indicating that unfucosylated RE cells may represent aberrant cellular behavior. We concluded that these patterns of lectin staining indicate a functional maturation as well as an integration of regenerating mucosa.

Positive chronotropic actions of parathyroid hormone and parathyroid hormone-related peptide are associated with increases in the current, I(f), and the slope of the pacemaker potential.

The classic calciotropic hormone parathyroid hormone (PTH) and its paracrine factor parathyroid hormone-related protein (PTHrP) both increase heart rate. We used standard electrophysiological techniques to study the effects of PTH and PTHrP on isolated rabbit sinus node, isolated canine Purkinje fibers, and disaggregated rabbit sinus node myocytes. Sinus node maximum diastolic potential, activation voltage, and amplitude were unchanged by PTH or PTHrP (P>.05). However, the slope of phase 4 and the automatic rate were increased at PTH and PTHrP > or = 10 nmol/L (P<.05). Comparable results were seen in canine Purkinje fibers. We then used the perforated-patch technique to study the I(f) pacemaker current in sinus node. PTH 12.5 nmol/L and PTHrP 12.5 to 18 nmol/L increased I(f) at -65 mV by 68+/-41% (n=5) and 69+/-50% (n=5), respectively. Actions of both agents were reversible. The increase in I(f) appeared to result from a change in maximal conductance and not a shift in the voltage dependence of activation. These observations provide, for the first time, direct electrophysiological support for the chronotropic actions of PTH and PTHrP. They suggest that classic hormones and paracrine factors can have multiple functions and that in the case of PTH and PTHrP, a newly recognized action is to alter automaticity directly.

Modulation of Ciliary Beat Frequency in the Upper Airway by Prostaglandins

Prostaglandins are ubiquitous within tissues and participate in several local regulatory functions. Previous reports have presented conflicting evidence on their role in ciliary activity in the upper airway. We studied the in vitro effects of prostaglandin E2(PGE2) and a prostaglandin I2analog (Iloprost) on the ciliary beat frequency (CBF) of human adenoid explants and rabbit sinus mucosa. Tissue explants were maintained in culture at 35°C. PGE2or Iloprost was added to the culture media at concentrations of 10–6, to 10–12M. CBF was determined using phase contrast microscopy and microphotometry. PGE2increased CBF with a maximum increase of 54.6% in rabbit mucosa (P &lt; 0.01) and 20.0% in human adenoid (P &lt; 0.009). Iloprost increased CBF with a maximum increase of 35.2% in rabbit mucosa (P &lt; 0.02) and 16.4% in human adenoid (P &lt; 0.03). Induced production of endogenous prostaglandins was determined by treating specimens with a cyclooxygenase inhibitor (diclofenac) prior to addition of PGE2or Iloprost. Diclofenac had no effect on the ciliostimulatory effects of PGE2(P &gt; 0.36), however it significantly decreased the ciliostimulatory effect of Iloprost (P &lt; 0.037). Both PGE2and Iloprost stimulate ciliary motility, yet the action of PGE2is direct, whereas Iloprost stimulates cilia by release of endogenous prostaglandins in both the human and rabbit.

Effects of potassium channel openers on Na + and K + currents in rabbit sinus node and atrial myocytes

The effects of the potassium channel openers (KCOs) Cromakalim and Lemakalim on rabbit sinoatrial and atrial myocytes were examined by means of the whole-cell patch-clamp technique. Lemakalim (up to 100 μM) had no effect on potassium current in sinoatrial cells. Both Lemakalim and Cromakalim (100 μM) displayed a two-fold action on atrial myocytes: (1) they increased an outwardly rectifying conductance at potentials positive to E K and, (2) they markedly decreased a TTX-sensitive Na + current active in the voltage range −50 −30 mV. This novel action on TTX-sensitive currents is of particular since these two benzopyrans have been thought to specifically target potassium channels.

Gap junctional channels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology.

The subcellular mechanism of cell-to-cell communication in the natural pacemaker region of the mammalian heart was studied using electrophysiological and immunofluorescence techniques in isolated pairs of rabbit sinus nodal cells. By measuring whole-cell currents using a double patch-clamp approach, it was demonstrated that communication in the sinus node is mediated through gap junctional channels similar to those in other types of adult cardiac cell pairs. Macroscopic sinus nodal junctional resistance had a mean value of 387.9 +/- 97.1 M omega (mean +/- SEM, n = 10) and was greatly increased by superfusion with alkanols. Single-channel junctional conductance could be resolved in three cell pairs. Given their high membrane resistance (1.16 +/- 0.32 G omega, n = 12), the electrical coupling provided by as few as three gap junctional channels between nodal cells will allow for pacemaker synchronization. Further evidence for the presence of the channels was obtained from immunofluorescent double-labeling of desmin and the gap junction protein (connexin43) in sinus nodal tissue as well as in cultured sinus nodal cells. Using antisera against residues 243-257 of the connexin43 protein, a specific staining at the site of cell-to-cell apposition was demonstrated. These data provide direct evidence in favor of electronic coupling as the means for achieving pacemaker synchronization in the rabbit sinus node.

Electrophysiological actions of a new antiarrhythmic drug, bisaramil, on isolated heart preparations

Electrophysiological effects of bisaramil--a new antiarrhythmic drug under clinical trial--were investigated on isolated heart preparations, at a concentration range of 2.3-23 x 10(-6) M. Bisaramil dose dependently decreased the maximum rate of depolarization (Vmax), action potential amplitude (APA) and overshoot (OS) both in auricle and in papillary muscle of guinea-pig heart. There was no significant and obvious effect on the duration of the action potential and the resting membrane potential was also unchanged. Bisaramil slowed the spontaneous frequency of pacemaker cells in rabbit sinus node preparation due to its inhibitory effect on slow diastolic depolarization (SDD). Bisaramil was able to inhibit slow Ca(2+)-action potentials induced by isoprenaline on K(+)-depolarized papillary muscle. Results obtained with transmembrane current measurements revealed that bisaramil inhibited both fast Na(+)-current and slow Ca(2+)-current in frog sinoauricular fibres at the same concentration. Bisaramil with a mixed mode of the action seems to be a very promising drug.

Morphological organization of the post-ganglionic sympathetic innervation of the rabbit's sinus node

Morphological organization of the post-ganglionic sympathetic innervation of the rabbit's sinus node

Phase resetting and entrainment of pacemaker activity in single sinus nodal cells.

The phase-resetting and entrainment properties of single pacemaker cells were studied using computer simulations in a model of the rabbit sinus nodal cell, as well as using the whole-cell patch-clamp (current-clamp) technique in isolated rabbit sinus nodal cells. Spontaneous electrical activity in the cell model was reconstructed using Hodgkin-Huxley-type equations describing time- and voltage-dependent membrane currents. In both simulations and experiments, single subthreshold current pulses (depolarizing or hyperpolarizing) were used to scan the spontaneous cycle of the cells. Such pulses perturbed the subsequent discharge, producing temporary phasic changes in pacemaker period, and enabled the construction of phase response curves. On the basis of these results, we studied entrainment characteristics of the cells. For example, application of repetitive pulses allowed for phasic changes in the spontaneous cycle and resulted in stable 1:1 entrainment at a range of basic cycle length around the spontaneous cycle, or a 2:1 pattern at basic cycle length values about half the spontaneous cycle length. Between the two entrainment zones, complex Wenckebach-like patterns (e.g., 5:4, 4:3, and 3:2) were observed. The experimental data from the isolated cell were further analyzed from a theoretical perspective, and the results showed that the topological characteristics of the phase-resetting behavior accounts for the experimentally observed patterns during repetitive stimulation of the cell. This first demonstration of phase resetting in single cells provides the basis for phenomena such as mutual entrainment between electrically coupled pacemaker cells, apparent intranodal conduction, and reflex vagal control of heart rate.

Electrical effects of okadaic acid extracted from black sponge on rabbit sinus node

1. Effects of okadaic acid on electrical responses and spontaneous activity in the dominant pacemaker cells of rabbit sinus node were investigated by use of microelectrode techniques. 2. Okadaic acid (10(-5) M to 4 x 10(-5) M) caused a shortening of cycle length of spontaneous firing (SPCL) accompanied by increases in both maximum upstroke velocity at phase 0 (Vmax) and amplitude of action potential. 3. All of the effects of okadaic acid were relatively well preserved in a low-Ca2+ medium (0.12 mM). Okadaic acid restored the spontaneous activity of sinus node pacemaker cells even in a Ca2(+)-deficient medium. 4. The effects of okadaic acid were markedly inhibited or abolished in a low Na+ medium (24 mM or 70 mM) and in the presence of a slow channel blocking agent, verapamil (10(-6) M). 5. In voltage-clamp experiments using a two-microelectrode technique, okadaic acid (10(-5) M) caused an increase in the slow inward current without affecting the outward current. At a higher concentration (4 x 10(-5) M), the drug increased the outward current. 6. These results indicate that okadaic acid causes an increase in spontaneous activity of sinus node pacemaker cells mediated by an enhancement of slow inward current (Isi) through verapamil-sensitive Ca2+ channels.