Proarrhythmia of azimilide and other class III antiarrhythmic agents in the adrenergically stimulated rabbit.

The ventricular proarrhythmic actions of five class III antiarrhythmic agents were compared in the Carlsson rabbit model. In adrenergically stimulated anesthetized rabbits, azimilide, clofilium, dofetilide, sematilide, and d,l-sotalol caused premature ventricular contractions and nonsustained and sustained ventricular tachyarrhythmias (NSVT and SVT) at pharmacologically equivalent intravenous doses that increased QTc intervals 20% (ED20). There were no significant differences between agents in the percentage of rabbits with serious arrhyhthmias at the ED20 doses of 5.2, 0.033, 0.015, 0.66, and 2.8 mg/kg i.v., respectively. Proarrhythmia was dose-dependent. Linear regression analysis of arrhythmia score versus log dose estimated the NSVT doses as 6.2, 0.055, 0.0089, 1.5, and 5.7, respectively. Analysis of arrhythmia states during a 10-min window after infusion when QTc prolongation was 20% showed that the compounds differed significantly in the proportion of time treated rabbits spent in SVT and combined NSVT and SVT. Rabbits treated with azimilide spent significantly less time in SVT and combined NSVT and SVT, followed in order of increasing time by d,l-sotalol, sematilide, clofilium, and dofetilide.

Effects of azimilide and d,l-sotalol on the heart rate and blood pressure response to isoproterenol in anesthetized rats.

The novel class III antiarrhythmic agent, azimilide, provides antifibrillatory protection in a rat model of ischemia-reperfusion arrhythmias. In other species azimilide's antifibrillatory mechanism is thought to be mediated predominantly through blockade of both the rapid and slow components of the delayed rectifier potassium current in ventricular myocytes. However, the delayed rectifier potassium current does not appear to control cardiac repolarization in the rat. One possible mechanism for antiarrhythmic efficacy in rats is the compound's beta-adrenergic blocking effect, previously seen in isolated guinea pig hearts. The purpose of this study was to evaluate the beta-adrenergic antagonistic effect of azimilide in the rat. Beta-adrenergic blockade was evaluated in the intact anesthetized rat by studying the effects of intravenous azimilide (at or above the antifibrillatory dose) and d,l-sotalol (a known beta-adrenergic antagonist) on heart rate and blood pressure responses to isoproterenol (0.14 Mg/kg i.v.). d,l-Sotalol (6.0 mg/kg) reduced (p < 0.05) the tachycardic response to isoproterenol from 133 +/- 11 to 80 +/- 10 beats/min, and 3.0 mg/kg of d,l-sotalol reduced the hypotensive response from -74 +/- 4 to -43 +/- 5 mmHg. Azimilide (5.0, 10.0, and 20.0 mg/kg) did not have a statistically significant effect on either the heart rate or blood pressure changes caused by isoproterenol. These data demonstrate that azimilide does not have a beta-adrenergic antagonist effect in the rat at antifibrillatory doses. Therefore, the antiarrhythmic effect of azimilide in the rat is mediated through a mechanism other than beta-blockade.

Influence of graft innervation on neovascularization of embryonic heart tissue grafted in oculo.

We have shown that sympathetic denervation increases subendocardial capillarity during left ventricular hypertrophy. To determine the direct effects of sympathetic innervation on development of the coronary microvasculature in the absence of hemodynamic load, we grafted avascular fetal rat atrial or ventricular tissue into the anterior eye chamber of host rats which had undergone unilateral superior cervical gangliectomies. Innervation to the contralateral eye chamber remained intact. The grafts were harvested 14 or 35 days later, and volume densities of blood vessels, myocytes, and extracellular matrix were determined using standard point-counting techniques on low-power electron micrographs. Graft perfusion and metabolism were assessed simultaneously with thallium-201 and 2-[14C]deoxy-D-glucose uptake, respectively. Innervation did not significantly alter the vascular volume densities or cellular composition of atrial or ventricular tissue compared with noninnervated tissue after either 14 or 35 days in oculo. Similarly, innervation did not significantly alter graft perfusion or metabolism. We conclude that sympathetic innervation does not directly influence the growth of the microvasculature or myocardial metabolism in hemodynamically unloaded, developing heart tissue.

Hepatic vein, hepatic parenchymal, and inferior vena caval mechanoreceptors with phrenic afferents.

Dogs were anesthetized with pentobarbital sodium and placed on positive-pressure ventilation. The right phrenic nerve and/or its C5 branch were prepared for afferent recording. The hepatic veins, hepatic parenchyma, diaphragm, and inferior vena cava were studied for mechanoreceptors using light pressure and stroking as the stimuli. Mechanosensitive areas were found in the hepatic veins, hepatic parenchyma of the right medial lobe, and inferior vena cava. The hepatic vein and inferior vena caval receptors are located in the same 1- to 2-cm region as the sphincters that are found in these vessels. This study presents the first experimental evidence for the existence of hepatic vein receptors, hepatic parenchymal receptors, and inferior vena caval mechanoreceptors with phrenic afferents in the dog. These sensory areas of the circulation may be involved in the neural control of venous return as well as mediating changes in intrahepatic and portal venous blood pressure during normal respiration.

Pericardial mechanoreceptors with phrenic afferents.

Pericardial mechanoreceptors with afferents in the phrenic nerves were studied in anesthetized dogs. The specific aims determined 1) if pericardial receptors with phrenic afferents exist in the dog; 2) the stimuli needed to activate these receptors; 3) the anatomic distribution of these pericardial receptors; and 4) which pericardial layer contains the receptors. Afferent activity was recorded from the phrenic nerves while the pericardium was probed. In 15 of 18 animals, pericardial receptors were found on the right side. In 12 of 18 animals pericardial receptors were located on the left side. Most of the mechanoreceptors were found in a band that paralleled the pericardiophrenic attachment, in the fibrous layer of the pericardium, overlying the atria and atrioventricular grooves. Some receptors had a cardiac rhythm, whereas others were stimulated by the inflating lung. None of the receptors were chemosensitive to capsaicin, bradykinin, or saline. This study is the first to demonstrate that the pericardium of the dog contains mechanosensitive receptors which are innervated by the phrenic nerve.

Autonomic influences on regional cardiac [14C]2-deoxyglucose uptake in paced dog heart.

Sixteen dogs were anesthetized with pentobarbital sodium and placed on positive pressure ventilation. Twelve animals were cardiac paced and four animals were non-paced controls. In four of the paced animals, the left cut peripheral end of the cervical vagus was stimulated electrically. In another four animals, the decentralized left stellate ganglion was stimulated efferently. The four remaining paced animals were controls. The animals were injected i.v. with a single bolus of [14C]2-deoxyglucose, 100 microCi/kg. After 45 minutes of periodic stimulation, the hearts were removed, sectioned and prepared for autoradiography. Cardiac pacing significantly (p less than 0.02) decreased systolic blood pressure, and significantly increased (p less than 0.05) glucose utilization by all regions of the heart, with the exception of the left ventricular endocardium and the left ventricular papillary muscles. Vagal efferent stimulation elicited significant (p less than 0.05) decreases in glucose utilization in both the right atrium and right ventricle. Conversely, efferent stellate ganglion stimulation had no effect on the glucose utilization of any regions of the myocardium studied. This study demonstrates that both cardiac pacing and cardiac vagal efferent neuronal stimulation can produce distinct changes in regional myocardial glucose utilization.

Regional myocardial glucose utilization by developing fetal and maternal hearts.

Regional glucose utilization of the developing fetal feline heart was assessed during three stages of gestation and compared with the maternal heart and non-pregnant controls. The specific aims were to determine: 1) if glucose utilization by the whole heart changes from early to late gestation; 2) if there are differences in glucose utilization by specific regions of the heart; 3) if these regional differences in glucose utilization are consistent throughout gestation. Regional myocardial glucose utilization was measured using the [14C] 2-deoxyglucose high spatial resolution autoradiographic technique. Eleven fetal and 16 adult hearts were studied. Two of the fetuses were at 49 days of gestation, three were at 35 days, and six were at 25 days of gestation. This was the first study to assess regional myocardial glucose utilization in the developing fetus. Glucose utilization by the fetal heart was greater than that seen in the normal control adult or maternal heart, and was highest during early gestation. The posterior wall of the left ventricle had glucose utilization twice that measured for the anterior wall. Other regions were not significantly different. This information indicates that availability of glucose to the fetus is important for normal cardiac metabolism and development.

Medullary sites mediating some abdominal vagal reflexes in the ferret using [14C]2-deoxyglucose.

The central projections of some abdominal visceral afferents passing through the vagal communicating branch were studied in anesthetized ferrets using [14C]2-deoxyglucose autoradiography. The reflex effects of electrical stimulation of the vagal communicating branch were studied while measurements of jejunal motor activity and transmural potential difference, a marker of electrogenic epithelial transport were made concurrently. The aim of this study was to examine brainstem projections of some afferent fibers in the communicating branch of the thoracic vagus nerve that are necessary for the reflex regulation of small intestinal motor activity and epithelial transport. In urethane-anesthetized ferrets, electrical stimulation of the cut central end of the vagal communicating branch increased jejunal motor activity and electrogenic epithelial transport. In addition, glucose utilization in the left medial sub-nucleus of the nucleus tractus solitarius and the dorsal motor nucleus of the vagus was significantly increased as compared with sham-operated non-stimulated control animals. Identical areas on the contralateral side of the brain showed no change in glucose utilization as compared with sham-operated non-stimulated controls. This functional brain-mapping study strongly suggests that the left medial sub-nucleus of the nucleus tractus solitarius and the dorsal motor nucleus of the vagus, in the ferret, are involved in processing alimentary afferent activity from both the small intestinal musculature and epithelium as well as the reflex changes in efferent vagal nerve activity to the same regions of the alimentary tract.

Fetal glucose utilization using maternal plasma glucose and scintillation values.

The hypothesis tested was that measurement of fetal plasma values for glucose and scintillation counts results in statistically the same calculated integrals and consequent values for cerebral glucose utilization as would be obtained if the maternal plasma values were used alone with the quantitative 2-[14C]deoxyglucose technique of Sokoloff et al. In 4 pregnant rabbits anesthetized with ketamine, a single bolus of 2-[14C]deoxyglucose was injected i.v. Fourteen periodic blood samples were taken from the adult rabbit and a single fetal placental unit simultaneously. The plasma integrals of the Sokoloff equation were calculated and compared statistically. The results demonstrate that there is no statistical difference between the maternal and fetal plasma integrals and therefore the fetal plasma values do not need to be measured for accurate measurements of fetal cerebral glucose utilization to be made using the 2-[14C]deoxyglucose technique of Sokoloff.

Cardiac, respiratory, and renal responses to stimulation of the external cuneate nucleus.

The cardiac, respiratory, and renal responses of electrical stimulation and microinjection of excitatory amino acids into the external cuneate nucleus were investigated in 57 cats anesthetized with pentobarbital sodium, paralyzed, and artificially ventilated. Trains of rectangular cathodal pulses of 40-100 microA at 50 Hz and 0.1 ms duration were delivered through monopolar glass microelectrodes with a tip diameter of 10-20 micron, filled with indium-Woods metal alloy. Electrical stimulation at 232 histologically identified sites within the external cuneate nucleus could evoke changes in arterial blood pressure, heart rate, and efferent renal sympathetic nerve activity. In a further set of experiments, a change in respiration was observed at 74 identified sites. An increase or decrease in all parameters measured could be elicited at different stimulus sites within the external cuneate nucleus. Repositioning of the electrode (0.2-0.4 mm) in depth or laterally could result in a different response with stimulation. Microinjections of D,L-homocysteic acid or glutamate could mimic the evoked changes in blood pressure, heart rate, efferent renal sympathetic nerve activity, and respiration. This suggests that the external cuneate nucleus contains cell bodies that may modulate components of various cardiac, respiratory and renal reflexes. It is proposed that the external cuneate nucleus may be involved in the integration of somato-autonomic reflex responses.

Alterations in cardiac [14C]deoxyglucose uptake induced by two renal afferent stimuli.

The reflex effects of renal afferents on the heart were studied in pentobarbital-anesthetized rats (400-425 g) using 2-[14C]deoxyglucose (DG). Three groups of rats were given a single bolus injection of DG (100 mu Ci/kg) 1) six controls, 2) four with periodic electrical stimulation of the proximal end of a cut renal nerve (2 Hz, 0.5-ms width) and 1-mA current, and 3) six with intermittent renal venous occlusion (unilateral). Forty-five minutes after injection the heart was removed, cooled quickly, and frozen-sectioned. Sections 20 micron thick were exposed to film for 12 days. The resulting autoradiographs were scanned using a computerized densitometer, and these densities were converted to relative glucose utilization (GlU, mumol X 100 g-1 X min-1) using the lumped constant for rat brain. Both renal venous occlusion and renal afferent nerve stimulation resulted in a decrease in blood pressure of 6.7 +/- 0.6 mmHg (P less than 0.001) and 7.3 +/- 0.7 mmHg (P less than 0.001) and heart rate-blood pressure product of 5.6 +/- 0.7% (P less than 0.001) and 8.8 +/- 0.8% (P less than 0.001), respectively, and afferent renal nerve stimulation induced a decrease in heart rate of 7.2 +/- 0.9 beats/min (P less than 0.01). However, when compared with control, renal venous occlusion induced a significant increase in GlU in left ventricular myocardium (LV myo, P less than 0.05), endocardium (LV endo, P less than 0.001), and papillary muscle (LV pap, P less than 0.001), whereas afferent renal nerve stimulation induced a significant increase in GlU in LV endo (P less than 0.05) and LV pap (P less than 0.002) only. This study shows both a reflex increase in GlU for the rat heart and a decrease in heart rate with either renal vein occlusion or afferent renal nerve stimulation.

Effect of arterial PCO2 on 2-[1-14C]deoxy-D-glucose uptake by feline cerebral arteries.

The effect of high and low arterial CO2 on the glucose utilization of nine major cerebral arteries was studied in cats anesthetized with pentothal using the quantitative 2-[1-14C]deoxy-D-glucose autoradiographic technique. All nine cerebral arteries from animals subjected to an arterial partial pressure of CO2 (PCO2) of 20 Torr utilized significantly more (P less than 0.025) glucose than the group subjected to an arterial PCO2 of 60 Torr. Mean relative glucose utilization of the 20-Torr PCO2 group was 105 +/- 9.5 mumol X 100g-1 X min-1 (+/- SE, n = 18) as compared with 49 +/- 6 mumol X 100g-1 X min-1 (+/- SE, n = 26) for the 60-Torr PCO2 group. This study demonstrates that blood vessels can be studied in vivo using the 2-[1-14C]deoxy-D-glucose autoradiographic technique. It also demonstrates that a physiological stimulus like CO2 can produce measurable changes in glucose utilization of cerebral arteries in vivo.

Spinal cord metabolic response to noxious radiant heat stimulation of the cat hind footpad.

The 2-deoxy[14C]glucose (2-DG) method was used to study glucose utilization in the cat lumbar spinal cord during noxious thermal stimulation to the hind footpad. Spinal cord glucose utilization in stimulated cats was twice that of control animals. Diffusely increased metabolic activity was seen in the ipsilateral dorsal horn. The highest levels of 2-DG were seen in the ipsilateral ventral horn, while a moderate increase was seen in the contralateral ventral horn.

Ventilation of a rat with a large-animal respirator.

Rats were effectively ventilated with 100% O2 mixed with room air utilizing a modified tracheostomy tube and a Bird Mark 7 respirator to maintain arterial blood gases within normal limits. A 3-cm segment of rubber pilot tubing was attached to a 15-mm respiratory connector and a 3-cm piece of polyethylene catheter tubing was fitted snugly into the other end. The catheter was inserted and secured into the trachea of 250- to 500-g Sprague-Dawley rats with the adaptor hose of the respirator fitted onto the 15-mm connector following tracheostomy. Manometer and inspiratory flow rate controls of the respirator were set to their minimum operating position. Appropriate rate control adjustments were made when necessary as determined by arterial blood gas measurements. By use of the above ventilation system, adequate arterial blood gases of anesthesized rats can be maintained for greater than 3 h.

The effects of morphine on sympathetic transmission in the stellate ganglion of the cat.

The aim of this study was to investigate which of the processes involved in synaptic transmission are affected by morphine in concentrations comparable to those used during surgical procedures. The effects of morphine sulfate on ganglionic transmission were studied in the stellate ganglion of the cat using intracellular and extracellular recordings in vitro. The neurons of the stellate ganglion were depolarized using preganglionic nerve stimulation, postganglionic nerve stimulation, and intracellular stimulation before and after introduction of morphine sulfate (up to 20 micrograms/mL). Tissue concentrations of morphine were estimated using radiolabeled morphine. Axonal transmission and the excitability of the postganglionic neurons to direct intracellular stimulation was not affected at the concentrations of morphine studied. In addition, morphine had a dose-dependent depolarizing effect on the resting membrane potential of most of the neurons in the stellate ganglion. Such neuronal depolarizations alone could initially produce excitation in some cell populations, followed by inhibition, secondary to the membrane depolarization, leading to depression of sympathetic nerve activity. The overall ganglionic transmission as recorded using an evoked potential was biphasic. At low doses morphine facilitated transmission, while at larger doses morphine attenuated evoked potentials. These effects do not appear to be mediated through classical opiate receptors since they are not blocked by naloxone.

Metabolic mapping of a cardiac reflex mediated by sympathetic ganglia in dogs.

The hypothesis tested was that some cardiac reflexes can be mediated by the neural mechanisms in thoracic sympathetic ganglia in addition to those existing in the spinal cord or brain stem. In mongrel dogs anesthetized with pentobarbital sodium the left stellate (SG) and middle cervical (MCG) ganglion were decentralized, and the left thoracic vagosympathetic trunk was cut cranial to the MCG and caudal to the MCG but above the heart. The left thoracic vagosympathetic trunk below the MCG was then stimulated afferently. A single bolus of 2-[14C]deoxyglucose was injected intravenously and the stimuli repeated periodically toward the decentralized ganglia for 45 min. The heart and ganglia were then removed, frozen, and sectioned for autoradiography. Significant increases in glucose utilization were found in both the SG and MCG compared with ganglia from nonstimulated control animals. Significant increases in the glucose utilization of the endocardial third of the left ventricle were also observed in the reflexly stimulated hearts. Neural mechanisms in the acutely decentralized SG and MCG could modify cardiac glucose utilization within specific areas of the heart. In addition, such ganglionic mechanisms may be concentrated within the caudal half of the SG. These data suggest that many neurons in the thoracic autonomic ganglia may be involved in local cardiac reflexes.

Vagal inhibition of respiratory-linked efferent activity in the carotid sinus nerve.

The hypothesis tested in this study was that glossopharyngeal efferent nerve activity coursing through the carotid sinus nerve has a central origin. Efferent activity in the carotid sinus nerve exhibited a respiratory rhythm in spontaneously breathing, closed-chest, mongrel dogs anesthetized with pentobarbital sodium (30 mg/kg iv). Carotid sinus nerve activity was recorded from the intact or cut central end of the carotid sinus nerve. Diaphragm electromyogram (D-EMG), carotid sinus pressure, systemic blood pressure, and electrocardiogram were also recorded. Before vagotomy, small increases in carotid sinus efferent nerve activity (CSENA) synchronous with increases in the D-EMG were observed during spontaneous inspiration. Section of the contralateral cervical vagosympathetic trunk markedly potentiated the increases in CSENA. Bilateral superior cervical ganglionectomy or nodose ganglionectomy failed to alter the increases in CSENA. Section of the ipsilateral glossopharyngeal nerve near the skull abolished the CSENA. This study demonstrates that respiratory-modulated glossopharyngeal efferents course through the carotid sinus nerve to the carotid sinus or carotid body. These efferents may be part of a central respiratory regulatory mechanism that may rapidly alter the sensitivity of the carotid sinus baroreceptors and/or carotid body receptors on a breath-to-breath basis.

Cardiovascular responses to noxious radiant heat in anesthetized cats.

The responses of heart rate and blood pressure to noxious radiant heat were studied in seven pentobarbital-anesthetized cats. Afferent activity recorded from the tibial nerve, systemic blood pressure, and heart rate were monitored as skin temperature of the hind footpad was raised to 53 degrees C for 20 s using radiant heat. The averaged tibial afferent nerve activity increased markedly as skin temperature approached 52 degrees C. Within 2-3 s of the onset of increased tibial nerve activity, systolic blood pressure increased an average of 32 mmHg and heart rate increased an average of 16 beats/min in the seven animals that were studied. The results of this study provide evidence for a somatosympathetic reflex that is initiated by cutaneous nociceptors. Under pentobarbital anesthesia, an increase in heart rate and blood pressure appears to be a reliable indicator of nociceptor activation.