Kyoung S. K. Chang

Propofol produces endothelium-independent vasodilation and may act as a Ca2+ channel blocker

The mechanism of vasodilation induced by propofol was investigated using isolated rat thoracic aortic rings. Aortic rings were precontracted with potassium chloride (KCl) (40 mM) or phenylephrine (PE) (3 × 10−8to 3 × 10−7M) in the presence and absence of intact endothelium. Propofol produced similar concentration-dependent relaxation in aortic rings with and without endothelium regardless of whether they were precontracted with KCl or PE. The relaxation response to propofol was significantly greater in KCl-contracted aortic rings than in PE-contracted aortic rings. The propofol concentration producing 50% relaxation from the contracted state (RC50) was lower in aortic rings contracted with KCl than with PE, both with (5 × 0.6 × 10−5M vs 8.3 × 5.7 × 10−4M, P < 0.001) and without intact endothelium (3.9 × 0.5 × 10−5M vs 7.2 × 3.8 × 10−4M, P < 0.001). Propofol inhibited the Ca2+-induced contractions of aortic rings exposed to Ca2+-free media and depolarized with KC1 (40 mM, 100 mM) in a dose-dependent manner. These effects are similar to those produced by verapamil. Propofol (5 × 10−5M) had minimal effect on the intracellular Ca2+release elicited by PE (10−5M). We conclude that vasodilation produced by propofol is not endothelium-dependent but is likely due to blockade of voltage-gated influx of extracellular Ca2+.

Midazolam produces vasodilation by mixed endothelium-dependent and -independent mechanisms.

Aortic rings were obtained from rat thoracic aorta and studied in vitro with and without functionally intact endothelium to determine whether “the mechanism” requires endothelium [or endothelium-derived relaxing factor (EDRF)]. In aortic rings precontracted with either phenylephrine (PE, 3 × 10−8−3 × 10−7 mol/L) or KC1 (40 mmol/L), midazolam produced concentration-dependent relaxation, with and without endothelium. Rings without endothelium demonstrated significantly less relaxation than those with endothelium regardless of whether they were precontracted with PE or KC1. With intact endothelium, midazolam produced greater relaxation in PE-contracted aortic rings than in KC1-contracted aortic rings; the midazolam concentration producing 50% relaxation from the contracted state (RC50) was 8.8 ± 3.6 ± 10−7 mol/L for PE-contracted rings and 3.3 ± 1.1 ± 10−6 mol/L for KC1-contracted rings (P < 0.05). In aortic rings with intact endothelium pretreated with NG-monomethyl-L-arginine (L-NMMA, 10−4 mol/ L), an inhibitor of nitric oxide (NO) synthesis, midazolam produced relaxation of similar magnitude to that seen in the denuded aortic rings except at the highest concentration (1 × 10−5 mol/L). Pretreatment with the cyclooxygenase inhibitor, indomethacin (2.5 × 10−5 mol/L), did not change the midazolam-induced relaxation in rings with intact endothelium as compared to untreated control aortic rings. In contrast to the intact endothelium state, when endothelium was removed, midazolam produced greater relaxation in the KC1-contracted aortic rings than in PE-contracted rings (RC50, 1.2 ± 0.3 ± 10−5 mol/L vs 2.3 ± 0.4 ± 10−5 mol/L, P < 0.05). Midazolam inhibited contractions produced by incremental addition of Ca2+ to aortic rings exposed to Ca2+-free, K+-depolarizing solution. In addition, midazolam inhibited contractions elicited by l,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoro-methyl)-phenyl]-3-pyridine carboxylic acid methyl ester (BAY K 8644), an activator of voltage-gated Ca2+ channels. These effects were similar to those produced by the Ca2+ channel blocker verapamil. Midazolam had minimal effect on intracellular Ca2+ release elicited by PE (10−5 mol/L), indicated by the observation that initial phasic contractions elicited by PE in a Ca2+-free media were similar in the presence and absence of midazolam. We conclude that midazolam produces vasodilation by endothelium-dependent and-independent mechanisms. Endothelium-dependent vasodilation produced by midazolam probably is mediated through the release of NO from endothelium. Endothelium-independent vasodilation seems to be linked to inhibition of voltage-gated Ca2+ channels.

Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABA(A) epsilon subunit.

BACKGROUND Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation. METHODS The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission. RESULTS Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents. CONCLUSIONS Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

Isoflurane Depresses Baroreflex Control of Heart Rate in Decerebrate Rats

Background Isoflurane inhibits baroreflex control of heart rate (HR) by poorly understood mechanisms. The authors examined whether suprapontine central nervous system cardiovascular regulatory sites are required for anesthetic depression. Methods The effects of isoflurane (1 and 2 rat minimum alveolar concentration [MAC]) on the baroreflex control of HR were determined in sham intact and midcollicular-transected decerebrate rats. Intravenous phenylephrine (0.2–12 &mgr;g/kg) and nitroprusside (1–60 &mgr;g/kg) were used to measure HR responses to peak changes in mean arterial pressure (MAP). Sigmoidal logistic curve fits to HR–MAP data assessed baroreflex sensitivity (HR/MAP), HR range, lower and upper HR plateau, and MAP at half the HR range (BP50). Four groups (two brain intact and two decerebrate) were studied before, during, and after isoflurane. To assess sympathetic and vagal contributions to HR baroreflex, &bgr;-adrenoceptor (1 mg/kg atenolol) or muscarinic (0.5 mg/kg methyl atropine) antagonists were administered systemically. Results Decerebration did not alter resting MAP and HR or baroreflex parameters. Isoflurane depressed baroreflex slope and HR range in brain-intact and decerebrate rats. In both groups, 1 MAC reduced HR range by depressing peak reflex tachycardia. Maximal reflex bradycardia during increases in blood pressure was relatively preserved. Atenolol during 1 MAC did not alter maximum reflex tachycardia. In contrast, atropine during 1 MAC fully blocked reflex bradycardia. Therefore, 1 MAC predominantly depresses sympathetic components of HR baroreflex. Isoflurane at 2 MAC depressed both HR plateaus and decreased BP50 in both groups. Conclusions Isoflurane depresses HR baroreflex control by actions that do not require suprapontine central nervous system sites. Isoflurane actions seem to inhibit HR baroreflex primarily by the sympathetic nervous system.

Ketamine differentially blocks sensory afferent synaptic transmission in medial nucleus tractus solitarius (mNTS).

Background Ketamine increases blood pressure and heart rate by unknown mechanisms, but studies suggest that an intact central nervous system and arterial baroreceptors are required. In the brain stem, medial nucleus tractus solitarius receives afferents from nodose neurons that initiate cardiovascular autonomic reflexes. Here, the authors assessed ketamine actions on afferent medial nucleus tractus solitarius synaptic transmission. Methods Ketamine was applied to horizontally sliced brain stems. Solitary tract (ST) stimulation evoked excitatory postsynaptic currents (eEPSCs) in medial nucleus tractus solitarius neurons. Capsaicin (200 nm) block of ST eEPSCs sorted neurons into sensitive (n = 19) and resistant (n = 23). In nodose ganglion slices, shocks to the peripheral vagal trunk activated afferent action potentials in sensory neurons classified by conduction velocities and capsaicin. Results Ketamine potently (10–100 &mgr;m) blocked small, ST-evoked N-methyl-d-aspartate synaptic currents found only in a subset of capsaicin-resistant neurons (6 of 12). Surprisingly, ketamine reversibly inhibited ST eEPSC amplitudes and induced synaptic failure at lower concentrations in capsaicin-sensitive than in capsaicin-resistant neurons (P < 0.005; n = 11 and 11). Spontaneous EPSCs using non–N-methyl-d-aspartate receptors were insensitive even to 1–3 mm ketamine, suggesting that ST responses were blocked presynaptically. Similarly, ketamine blocked C-type action potential conduction at lower concentrations than A-type nodose sensory neurons. Conclusion The authors conclude that ketamine inhibits postsynaptic N-methyl-d-aspartate receptors and presynaptic afferent processes in medial nucleus tractus solitarius. Unexpectedly, capsaicin-sensitive (C-type), unmyelinated afferents are significantly more susceptible to block than capsaicin-resistant (A-type), myelinated afferents. This differentiation may be related to tetrodotoxin-resistant sodium currents. Since C-type afferents mediate powerful arterial baroreflexes effects, these differential actions may contribute to ketamine-induced cardiovascular dysfunction.

Ketamine inhibits presynaptic and postsynaptic nicotinic excitation of identified cardiac parasympathetic neurons in nucleus ambiguus.

Background Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated possible central nervous system actions of ketamine to inhibit cardiac parasympathetic neurons in the brainstem by inhibiting multiple nicotinic excitatory mechanisms. Methods The authors used a novel in vitro approach to study the effect of ketamine on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording. The effects of ketamine were tested on nicotine-evoked ligand-gated currents and spontaneous glutamatergic miniature synaptic currents (mini) in cardiac parasympathetic preganglionic neurons. Results Ketamine (10 &mgr;m) inhibited (1) the nicotine (1 &mgr;m)-evoked presynaptic facilitation of glutamate release (mini frequency, 18 ± 7% of control; n = 9), and (2) the direct postsynaptic ligand-gated current (27 ± 8% of control; n = 9), but ketamine did not alter the amplitude of postsynaptic miniature non–N-methyl-d-aspartate currents. &agr; Bungarotoxin, an antagonist of &agr;7 containing nicotinic presynaptic receptors, blocked ketamine actions on mini frequency (n = 10) but not mini amplitude. Conclusions Ketamine inhibits the presynaptic nicotinic receptors responsible for facilitating neurotransmitter release, as well as the direct ligand-gated inward current, but does not alter the nicotinic augmentation of non–N-methyl-d-aspartate currents in brainstem parasympathetic cardiac neurons. Such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

Ketamine inhibits sodium currents in identified cardiac parasympathetic neurons in nucleus ambiguus.

Background Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated the possibility that ketamine increases heart rate by inhibiting the central cardiac parasympathetic mechanisms. Methods We used a novel in vitro approach to study the effect of ketamine on the identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording, and ketamine effects on isolated potassium (K+) and sodium (Na+) currents were studied. Results Cardiac nucleus ambiguus neurons (n = 14) were inherently silent, but depolarization evoked sustained action potential trains with little delay or adaptation. Ketamine (10 &mgr;m) reduced this response but had no effect on the voltage threshold for action potentials (n = 14;P > 0.05). The current–voltage relations for the transient K+ current and the delayed rectified K+ current (n = 5) were unaltered by ketamine (10 &mgr;m–1 mm). Ketamine depressed the total Na+ current dose-dependently (10 &mgr;m–1 mm). In addition, ketamine shifted the Na+ current inactivation curves to more negative potentials, thus suggesting the enhancement of the Na+ channel inactivation (P < 0.05; n = 7). In the presence of Cd2+, ketamine (10 &mgr;m) continued to inhibit voltage-gated Na+ currents, which recovered completely within 10 min. Conclusions Ketamine inhibits Na+ but not K+ channel function in brainstem parasympathetic cardiac neurons, and such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

Indigo Carmine Inhibits Endothelium-Dependent and -Independent Vasodilation

To investigate the potential mechanisms by which indigo carmine produces hypertension, we tested the hypothesis that indigo carmine inhibits endothelium-dependent vasodilation and determined the possible site of the inhibition (endothelium versus smooth muscle). Using isolated rat thoracic aortic rings that were precontracted with phenylephrine, we examined vasodilatory responses to acetylcholine, histamine, and Ca2+ ionophore A23187 (in endothelium-intact rings) and sodium nitroprusside and isoproterenol (in endothelium-denuded rings) in the presence and absence of indigo carmine. In addition, the effects of methylene blue on the acetylcholine- and sodium nitroprusside-induced vasodilation were compared with those of indigo carmine. Indigo carmine (10(-6), 10(-5), and 10(-4) mol/L) significantly inhibited receptor- and non-receptor-mediated endothelium-dependent vasorelaxation. Indigo carmine (10(-4) mol/L) also inhibited endothelium-independent vasorelaxation induced by sodium nitroprusside (an activator of vascular smooth muscle soluble guanylyl cyclase), although to a lesser extent than vasodilation from acetylcholine, histamine, and Ca2+ ionophore A23187. In contrast, indigo carmine (10(-4) mol/L) had no effect on the vasodilation induced by isoproterenol (an activator of adenylyl cyclase), indicating that indigo carmine selectively inhibits nitric oxide-mediated responses. Methylene blue, a known inhibitor of soluble guanylyl cyclase, inhibited both acetylcholine- and sodium nitroprusside-induced vasorelaxation. The inhibition was also greater in the acetylcholine- than the sodium nitroprusside-induced vasodilation. These results suggest that indigo carmine, like methylene blue, may inhibit endothelium-dependent relaxation by a mechanism that involves two levels. The major action of indigo carmine appears to be at the level of nitric oxide generation and/or release from the endothelial cell. In addition, indigo carmine appears to inhibit vascular smooth muscle guanylyl cyclase. Thus, indigo carmine may elevate blood pressure by interfering with these nitric oxide-mediated vasodilatory mechanisms.

Bupivacaine Inhibits Baroreflex Control of Heart Rate in Conscious Rats

Background Because exposure to intravenously administered bupivacaine may alter cardiovascular reflexes, the authors examined bupivacaine actions on baroreflex control of heart rate in conscious rats. Methods Baroreflex sensitivity (pulse interval vs. systolic blood pressure in ms/mmHg) was determined before, and 1.5 and 15.0 min after rapid intravenous administration of bupivacaine (0.5, 1.0, and 2.0 mg/kg) using heart rate changes evoked by intravenously administered phenylephrine or nitroprusside. The actions on the sympathetic and parasympathetic autonomic divisions of the baroreflex were tested in the presence of a muscarinic antagonist methyl atropine and a &bgr;-adrenergic antagonist atenolol. Results Within seconds of injection of bupivacaine, mean arterial pressure increased and heart rate decreased in a dose-dependent manner. Baroreflex sensitivity was unaltered after administration of 0.5 mg/kg bupivacaine. In addition, 1 mg/kg bupivacaine at 1.5 min depressed phenylephrine-evoked reflex bradycardia (0.776 ± 0.325 vs. 0.543 ± 0.282 ms/mmHg, P < 0.05) but had no effect on nitroprusside-induced tachycardia. Bupivacaine (2 mg/kg), however, depressed reflex bradycardia and tachycardia (phenylephrine, 0.751 ± 0.318 vs. 0.451 ± 0.265; nitroprusside, 0.839 ± 0.256 vs. 0.564 ± 0.19 ms/mmHg, P < 0.05). Baroreflex sensitivity returned to prebupivacaine levels by 15 min. Bupivacaine (2 mg/kg), in the presence of atenolol, depressed baroreflex sensitivity (phenylephrine, 0.633 ± 0.204 vs. 0.277 ± 0.282; nitroprusside, 0.653 ± 0.142 vs. 0.320 ± 0.299 ms/mmHg, P < 0.05). In contrast, bupivacaine did not alter baroreflex sensitivity in the presence of methyl atropine. Conclusions Bupivacaine, in clinically relevant concentrations, inhibits baroreflex control of heart rate in conscious rats. This inhibition appears to involve primarily vagal components of the baroreflex–heart rate pathways.

Clinically relevant concentrations of bupivacaine inhibit rat aortic baroreceptors

Bupivacaine is clinically associated with cardiovascular toxicity. To examine the possible role of drug actions at arterial baroreceptors, we studied discharge properties of baroreceptors in an in vitro aortic nerve-aortic arch preparation from rats. We measured single fiber discharge, pressure, and aortic diameter simultaneously during perfusion of the aortic arch with bupivacaine. Perfusion mean arterial pressure was held at 80 mm Hg. Only regularly discharging, presumably myelinated, baroreceptors were studied. To assess pressure threshold, threshold frequency, and maximum discharge rate, nerve activity was evoked by slow ramps of increasing pressure (< 2 mm Hg/s) beginning at 20 mm Hg and ranging up to 150-170 mm Hg. Following replicate control measurements, test ramps were repeated in the presence of sodium nitroprusside (1 microM) and phentolamine (1 microM) to eliminate potential smooth muscle and alpha 1-adrenoceptor effects, respectively. Bupivacaine was then added to the perfusate in increasing concentrations from 0.1 to 50 microM for 15 min to construct a full concentration-response curve at each level. Individual baroreceptors showed substantial depression of maximum discharge frequency and/or increases in pressure threshold at 1-5 microM bupivacaine. In overall population averages (n = 7), 5-10 microM bupivacaine clearly reduced maximum discharge and shifted the pressure threshold to higher values (P < 0.01). The net result was a general depression of discharge. Concentrations as low as 10 microM bupivacaine completely blocked discharge in some baroreceptors. Inasmuch as the pressure-diameter relations were not changed, discharge relations plotted against diameter showed equivalent changes. Bupivacaine-free solution reversed the block in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)