Mitochondrial inner membrane electrophysiology assessed by rhodamine-123 transport and fluorescence.

Rhodamine-123 is widely used to make dynamic measurements of mitochondrial membrane potential both in vitro and in situ. Yet data interpretation is difficult due to a lack of quantitative understanding of how membrane potential and measured fluorescence are related. To develop such understanding, a model for dye transport across the mitochondrial inner membrane and partition into the membrane was developed. The model accounts for experimentally measured dye self-quenching and was integrated into a model of mitochondrial electrophysiology to estimate transients in mitochondrial membrane potential from kinetic fluorescence measurements. Our analysis indicates that (i) R123 fluorescence peaks at concentrations near 50 microM due to self-quenching; (ii) measured fluorescence intensity and membrane potential are related by a non-linear calibration curve sensitive to certain experimental details, including total concentration of dye and mitochondria in suspensions; and (iii) the time courses of membrane potential and electron transport fluxes following a perturbation (i.e. addition of ADP) significantly differ from observed transients in fluorescence intensity. These findings are consistent with the model predictions that mitochondria display a characteristic time of response to changes in substrate concentration of less than 0.1 s, corresponding to the time scale over which the rate of ATP synthesis changes to meet changes in ADP concentration.

Comparison of hyperkalemic cardioplegia with altered [CaCl2] and [MgCl2] on [Ca2+]i transients and function after warm global ischemia in isolated hearts.

AIM: [MgCl(2)] and [CaCl(2)] may modify the cardioprotective effects of hyperkalemic cardioplegia (CP). We changed [MgCl(2)] and [CaCl(2)] in a CP solution to examine their effects on [Ca(2+)]i transients and cardiac function before and after global normothermic ischemia. METHODS: After stabilization and loading of indo 1-AM in Kreb's solution (KR), each heart was perfused with either KR or 1 of 4 CP solutions before 37 degrees C, 30 min ischemia followed by reperfusion with KR. The KR solution contained, in mM, 4.5 KCl, 2.4 MgCl(2) and 2.5 CaCl(2); the CP solutions had in addition to 18 KCl: CP 1 (control CP): 2.4 MgCl(2), 2.5 CaCl(2); CP 2: 7.2 MgCl(2), 2.5 CaCl(2); CP 3, 7.2 MgCl(2), 1.25 CaCl(2); CP 4: 2.4 MgCl(2), 1.25 CaCl(2). RESULTS: In the KR group [Ca(2+)]i markedly increased on early reperfusion while functional return (LVP, dLVP/dt((max and min))) was much reduced; each CP group led to reduced [Ca(2+)]i loading and improved function. The rates of cytosolic Ca(2+) fluxes (d[Ca(2+)]/dt(max) and d[Ca(2+)]/dt(min)) increased significantly compared to baseline in the KR group, but were mostly suppressed in the CP groups, and d[Ca(2+)]/dt(min) was lower after CP 4 compared to CP 1 on reperfusion. At 60 min reperfusion, LVP area to [Ca(2+)] area and cardiac efficiency to phasic [Ca(2+)] relationships were shifted after KR, but not after CP 1-4. With similar functional recovery, [Ca(2+)] transient and [Ca(2+)] area were significantly lower after CP 4 than after CP 1. CONCLUSION: Increasing [MgCl(2)] (CP 2 and 3) did not improve cardiac function or reduce Ca(2+) transients on reperfusion better than the other CP groups, but reducing [CaCl(2)] (CP 3 and 4) was more effective in reducing [Ca(2+)] transients on reperfusion after global ischemia.

Blocking Na(+)/H(+) exchange reduces [Na(+)](i) and [Ca(2+)](i) load after ischemia and improves function in intact hearts.

We determined in intact hearts whether inhibition of Na(+)/H(+) exchange (NHE) decreases intracellular Na(+) and Ca(2+) during ischemia and reperfusion, improves function during reperfusion, and reduces infarct size. Guinea pig isolated hearts were perfused with Krebs-Ringer solution at 37 degrees C. Left ventricular (LV) free wall intracellular Na(+) concentration ([Na(+)](i)) and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured using fluorescence dyes. Hearts were exposed to 30 min of ischemia with or without 10 microM of benzamide (BIIB-513), a selective NHE-1 inhibitor, infused for 10 min just before ischemia or for 10 min immediately on reperfusion. At 2 min of reperfusion, BIIB-513 given before ischemia decreased peak increases in [Na(+)](i) and [Ca(2+)](i), respectively, from 2.5 and 2.3 times (controls) to 1.6 and 1.3 times pre-ischemia values. At 30 min of reperfusion, BIIB-513 increased systolic-diastolic LV pressure (LVP) from 49 +/- 2% (controls) to 80 +/- 2% of pre-ischemia values. BIIB-513 reduced ventricular fibrillation by 54% and reduced infarct size from 64 +/- 1% to 20 +/- 3%. First derivative of the LVP, O(2) consumption, and cardiac efficiency were also improved by BIIB-513. Similar results were obtained with BIIB-513 given on reperfusion. These data show that Na(+) loading is a marker of reperfusion injury in intact hearts in that inhibiting NHE reduces Na(+) and Ca(2+) loading during reperfusion while improving function. These results clearly implicate the ionic basis by which inhibiting NHE protects the guinea pig intact heart from ischemia-reperfusion injury.

Ischemic and anesthetic preconditioning reduces cytosolic [Ca2+] and improves Ca(2+) responses in intact hearts.

Ca(+) loading during reperfusion after myocardial ischemia is linked to reduced cardiac function. Like ischemic preconditioning (IPC), a volatile anesthetic given briefly before ischemia can reduce reperfusion injury. We determined whether IPC and sevoflurane preconditioning (SPC) before ischemia equivalently improve mechanical and metabolic function, reduce cytosolic Ca(2+) loading, and improve myocardial Ca(2+) responsiveness. Four groups of guinea pig isolated hearts were perfused: no ischemia, no treatment before 30-min global ischemia and 60-min reperfusion (control), IPC (two 2-min occlusions) before ischemia, and SPC (3.5 vol%, two 2-min exposures) before ischemia. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured at the left ventricular (LV) free wall with the fluorescent probe indo 1. Ca(2+) responsiveness was assessed by changing extracellular [Ca(2+)]. In control hearts, initial reperfusion increased diastolic [Ca(2+)] and diastolic LV pressure (LVP), and the maximal and minimal derivatives of LVP (dLVP/dt(max) and dLVP/dt(min), respectively), O(2) consumption, and cardiac efficiency (CE). Throughout reperfusion, IPC and SPC similarly reduced ischemic contracture, ventricular fibrillation, and enzyme release, attenuated rises in systolic and diastolic [Ca(2+)], improved contractile and relaxation indexes, O(2) consumption, and CE, and reduced infarct size. Diastolic [Ca(2+)] at 50% dLVP/dt(min) was right shifted by 32-53 +/- 8 nM after 30-min reperfusion for all groups. Phasic [Ca(2+)] at 50% dLVP/dt(max) was not altered in control but was left shifted by -235 +/- 40 nM [Ca(2+)] after IPC and by -135 +/- 20 nM [Ca(2+)] after SPC. Both SPC and IPC similarly reduce Ca(2+) loading, while augmenting contractile responsiveness to Ca(2+), improving postischemia cardiac function and attenuating permanent damage.

Volatile anaesthetics restore bradykinin and serotonin-induced coronary vasodilation after blocking nitric oxide synthase: lack of anaesthetic effects on KATP channels and prostaglandin pathways.

BACKGROUND AND OBJECTIVE: Volatile anaesthetic effects on altering tone after blocking nitric oxide synthase, cyclo-oxygenase-prostaglandin synthase and KATP channel pathways are controversial. We examined in isolated guinea pig hearts whether anaesthetics alter bradykinin and 5-hydroxytryptamine-induced effects on coronary flow and percentage oxygen extraction after blocking these pathways. METHODS: Before and during exposure to sevoflurane, halothane or isoflurane, hearts were infused with 10-13-10-8 M bradykinin, or 10-8-10-6 M 5-hydroxytryptamine (serotonin), with either L-NAME, indomethacin, or glibenclamide. Bradykinin or 5-hydroxytryptamine alone increased flow and decreased percentage oxygen extraction in a concentration-dependent manner; these effects were largely blocked by L-NAME (nitro-L-arginine methylester), which also decreased basal flow and increased basal percentage oxygen extraction. RESULTS: The anaesthetics restored bradykinin and 5-hydroxytryptamine-induced increases in flow or decreases in percentage oxygen extraction after inhibition by L-NAME. Indomethacin or glibenclamide alone had little effect on basal flow and percentage oxygen extraction. The anaesthetics restored bradykinin and 5-hydroxytryptamine-induced increases in flow or decreases in percentage oxygen extraction after inhibition by L-NAME. Indomethacin or glibenclamide alone had little effect on basal flow and percentage oxygen extraction. Drug-induced increases in flow and decreases in percentage oxygen extraction in the absence or presence of glibenclamide or indomethacin were not altered at either of the two concentrations of anaesthetics. CONCLUSIONS: Endothelium-dependent vasodilatation is not affected by blocking prostaglandin release or KATP channels in the intact heart even in the presence of an anaesthetic. However, the diminished responses to vasodilators after nitric oxide synthase inhibition is largely restored or enhanced by anaesthetics.

Changes in [Na(+)](i), compartmental [Ca(2+)], and NADH with dysfunction after global ischemia in intact hearts.

We measured the effects of global ischemia and reperfusion on intracellular Na(+), NADH, cytosolic and mitochondrial (subscript mito) Ca(2+), relaxation, metabolism, contractility, and Ca(2+) sensitivity in the intact heart. Langendorff-prepared guinea pig hearts were crystalloid perfused, and the left ventricular (LV) pressure (LVP), first derivative of LVP (LV dP/dt), coronary flow, and O(2) extraction and consumption were measured before, during, and after 30-min global ischemia and 60-min reperfusion. Ca(2+), Na(+), and NADH were measured by luminescence spectrophotometry at the LV free wall using indo 1 and sodium benzofuran isophthalate, respectively, after subtracting changes in tissue autofluorescence (NADH). Mitochondrial Ca(2+) was assessed by quenching cytosolic indo 1 with MnCl(2). Mechanical responses to changes in cytosolic-systolic (subscript sys), diastolic (subscript dia), and mitochondrial Ca(2+) were tested over a range of extracellular [Ca(2+)] before and after ischemia-reperfusion. Both [Ca(2+)](sys) and [Ca(2+)](dia) doubled at 1-min reperfusion but returned to preischemia values within 10 min, whereas [Ca(2+)](mito) was elevated over 60-min reperfusion. Reperfusion dissociated [Ca(2+)](dia) and [Ca(2+)](sys) from contractile function as LVP(sys-dia) and the rise in LV dP/dt (LV dP/dt(max)) were depressed by one-third and the fall in LV dP/dt (LV dP/dt(min)) was depressed by one-half at 30-min reperfusion, whereas LVP(dia) remained markedly elevated. [Ca(2+)](sys-dia) sensitivity at 100% LV dP/dt(max) was not altered after reperfusion, but [Ca(2+)](dia) at 100% LV dP/dt(min) and [Ca(2+)](mito) at 100% LV dP/dt(max) were markedly shifted right on reperfusion (ED(50) +36 and +125 nM [Ca(2+)], respectively) with no change in slope. NADH doubled during ischemia but returned to normal on initial reperfusion. The intracellular [Na(+)] ([Na(+)](i)) increased minimally during ischemia but doubled on reperfusion and remained elevated at 60-min reperfusion. Thus Na(+) and Ca(2+) temporally accumulate during initial reperfusion, and cytosolic Ca(2+) returns toward normal, whereas [Na(+)](i) and [Ca(2+)](mito) remain elevated on later reperfusion. Na(+) loading likely contributes to Ca(2+) overload and contractile dysfunction during reperfusion.

Reduced cytosolic Ca(2+) loading and improved cardiac function after cardioplegic cold storage of guinea pig isolated hearts.

BACKGROUND: Hypothermia is cardioprotective, but it causes Ca(2+) loading and reduced function on rewarming. The aim was to associate changes in cytosolic Ca(2+) with function in intact hearts before, during, and after cold storage with or without cardioplegia (CP). METHODS AND RESULTS: Guinea pig hearts were initially perfused at 37 degrees C with Krebs-Ringer's (KR) solution (in mmol/L: Ca(2+) 2.5, K(+) 5, Mg(2+) 2.4). One group was perfused with CP solution (Ca(2+) 2.5, K(+) 18, Mg(2+) 7.2) during cooling and storage at 3 degrees C for 4 hours; another was perfused with KR. LV pressure (LVP), dP/dt, O(2) consumption, and cardiac efficiency were monitored. Cytosolic phasic [Ca(2+)] was calculated from indo 1 fluorescence signals obtained at the LV free wall. Cooling with KR increased diastolic and phasic [Ca(2+)], whereas cooling with CP suppressed phasic [Ca(2+)] and reduced the rise in diastolic [Ca(2+)]. Reperfusion with warm KR increased phasic [Ca(2+)] 86% more after CP at 20 minutes and did not increase diastolic [Ca(2+)] at 60 minutes, compared with a 20% increase in phasic [Ca(2+)] after KR. During early and later reperfusion after CP, there was a 126% and 50% better return of LVP than after KR; during later reperfusion, O(2) consumption was 23% higher and cardiac efficiency was 38% higher after CP than after KR. CONCLUSIONS: CP decreases the rise in cardiac diastolic [Ca(2+)] observed during cold storage in KR. Decreased diastolic [Ca(2+)] and increased systolic [Ca(2+)] after CP improves function on reperfusion because of reduced Ca(2+) loading during and immediately after cold CP storage.

Enhanced contractile responsiveness to cytosolic Ca(2+) by delta-2 opioid agonist deltorphin in intact guinea pig hearts.

Opioid receptor subtypes, delta and kappa, are found in cardiac tissue and may play a role in cardiac function. We explored if the synthetic opioid delta(2)[D-Ala(2)]-deltorphin (DTP) and mu peptide agonist [D-Ala(2)]-enkephalin (DAMGO) alter the left ventricular pressure (LVP) [Ca(2+)](i) relationship in isolated guinea pig hearts. LV phasic [Ca(2+)](i) was measured from dual fluorescence signals using indo 1. Ca(2+) transients were corrected and calibrated to nM [Ca(2+)](i). Diastolic (d), systolic (s) [Ca(2+)](i), and s-d[Ca(2+)](i) were plotted v LVP at 0.3 to 6.8 mM [CaCl(2)](e)to assess the association of contractility to Ca(2+). Also given were naltriben (NTB) and CTOP, delta(2) and mu antagonists, and nifedipine (NIF) and thapsigargin (THAP). From a control of 880+/-95 nM (SEM), DTP decreased s-d[Ca(2+)](max) to 525+/-82 nM after DTP and to 405+/-84 nM after NIF, whereas THAP increased s-d[Ca(2+)](max)to 1605+/-275 nM. NTB, 795+/-33 nM, NTB+DTP, 820+/-98 nM, DAMGO, 970+/-82 nM, and DAMGO+CTOP, 830+/-93 nM, gave values similar to controls. From a control value of 61+/-4 mm Hg, LVP(max)was increased by DTP to 73+/-3 mmHg and by THAP to 77+/-2 mmHg, was unchanged by DAMGO at 48+/-6 mmHg, and was decreased by NIF to 24+/-2 mmHg. Compared to the control value of 594+/-18 nM, less s-d[Ca(2+)](i) was required to attain 50% s-dLVP(max)(curve left shift) with increasing [CaCl(2)](e) for DTP, 407+/-17 nM, and more was required for THAP, 737+/-35 nM. DTP raised the slope max of s-dLVP(max)(100%) v. s-d[Ca(2+)](i)by 2.7-fold. This indicates DTP enhances cardiac performance by enhancing responsiveness to cytosolic Ca(2+)rather than by raising diastolic Ca(2+) and subsequently released Ca(2+), as does THAP.

Xenon does not alter cardiac function or major cation currents in isolated guinea pig hearts or myocytes.

BACKGROUND: The noble gas xenon (Xe) has been used as an inhalational anesthetic agent in clinical trials with little or no physiologic side effects. Like nitrous oxide, Xe is believed to exert minimal unwanted cardiovascular effects, and like nitrous oxide, the vapor concentration to achieve 1 minimum alveolar concentration (MAC) for Xe in humans is high, i.e., 70-80%. In the current study, concentrations of up to 80% Xe were examined for possible myocardial effects in isolated, erythrocyte-perfused guinea pig hearts and for possible effects on altering major cation currents in isolated guinea pig cardiomyocytes. METHODS: Isolated guinea pigs hearts were perfused at 70 mm Hg via the Langendorff technique initially with a salt solution at 37 degrees C. Hearts were then perfused with fresh filtered (40-microm pore) and washed canine erythrocytes diluted in the salt solution equilibrated with 20% O2 in nitrogen (control), with 20% O2, 40% Xe, and 40% N2, (0.5 MAC), or with 20% O2 and 80% Xe (1 MAC), respectively. Hearts were perfused with 80% Xe for 15 min, and bradykinin was injected into the blood perfusate to test endothelium-dependent vasodilatory responses. Using the whole-cell patch-clamp technique, 80% Xe was tested for effects on the cardiac ion currents, the Na+, the L-type Ca2+, and the inward-rectifier K+ channel, in guinea pig myocytes suffused with a salt solution equilibrated with the same combinations of Xe, oxygen, and nitrogen as above. RESULTS: In isolated hearts, heart rate, atrioventricular conduction time, left ventricular pressure, coronary flow, oxygen extraction, oxygen consumption, cardiac efficiency, and flow responses to bradykinin were not significantly (repeated measures analysis of variance, P>0.05) altered by 40% or 80% Xe compared with controls. In isolated cardiomyocytes, the amplitudes of the Na+, the L-type Ca2+, and the inward-rectifier K+ channel over a range of voltages also were not altered by 80% Xe compared with controls. CONCLUSIONS: Unlike hydrocarbon-based gaseous anesthetics, Xe does not significantly alter any measured electrical, mechanical, or metabolic factors, or the nitric oxide-dependent flow response in isolated hearts, at least partly because Xe does not alter the major cation currents as shown here for cardiac myocytes. The authors' results indicate that Xe, at approximately 1 MAC for humans, has no physiologically important effects on the guinea pig heart.

Modulation of myocardial function and [Ca2+] sensitivity by moderate hypothermia in guinea pig isolated hearts.

Cardiac hypothermia alters contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]i over a range of extracellular CaCl2 concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37 degrees C, 27 degrees C, and 17 degrees C. Transmural LV phasic [Ca2+] was measured using the Ca2+ indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+ released per beat (area Ca2+) were plotted as a function of 0.3-4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/dt) and relaxation (-dLVP/dt), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]i and slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]e was shifted upward at 17 degrees C and 27 degrees C, whereas that of phasic [Ca2+]) to [CaCl2]e was shifted upward at 17 degrees C but not at 27 degrees C. The relationships of phasic [Ca2+]i to developed LVP, +dLVP/dt, and LVP(area) were progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+ with little effect on systolic Ca2+ or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+ and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.

Airway obstruction due to late-onset angioneurotic edema from angiotensin-converting enzyme inhibition.

PURPOSE: Angioneurotic edema is a well-documented complication of angiotensin-converting enzyme inhibitors (ACEI). We report a case of acute airway obstruction from a late-onset, probable ACEI-related angioneurotic edema and its subsequent management. CLINICAL FEATURES: A 48-yr-old obese man presented for transurethral resection of a bladder tumour (TURBT). His past medical history included hypertension controlled with hydrochlorothiazide and quinapril which had been started 13 mo earlier. Previous surgery was uncomplicated. Midazolam was used for premedication and for intraoperative sedation together with fentanyl and propofol. After uneventful spinal anesthesia with bupivacaine, operation and recovery, he was transferred to the floor. Five hours later he developed severe edema of his face, tongue and neck, with drooling, that progressed into airway obstruction and respiratory arrest. Ventilation was restored via immediate cricothyroidotomy, and a subsequent tracheotomy was completed uneventfully in the operating room. His serum C1 esterase inhibitor levels at 1, 5 and 23 days later were normal. The angioneurotic edema was attributed to the ACEI treatment. The edema resolved after 48 hr, and further follow-up was unremarkable. CONCLUSION: This observation is consistent with other reports that angioneurotic edema from ACEI can occur many months after the initiation of treatment. This can involve the airway and may produce life-threatening respiratory compromise. Physicians should be aware of this association and the possible need for immediate surgical intervention for the establishment of an airway in case of worsening edema or respiratory arrest.

Sevoflurane mimics ischemic preconditioning effects on coronary flow and nitric oxide release in isolated hearts.

BACKGROUND: Like ischemic preconditioning, certain volatile anesthetics have been shown to reduce the magnitude of ischemia/ reperfusion injury via activation of K+ adenosine triphosphate (ATP)-sensitive (K(ATP)) channels. The purpose of this study was (1) to determine if ischemic preconditioning (IPC) and sevoflurane preconditioning (SPC) increase nitric oxide release and improve coronary vascular function, as well as mechanical and electrical function, if given for only brief intervals before global ischemia of isolated hearts; and (2) to determine if K(ATP) channel antagonism by glibenclamide (GLB) blunts the cardioprotective effects of IPC and SPC. METHODS: Guinea pig hearts were isolated and perfused with Krebs-Ringer's solution at 55 mm Hg and randomly assigned to one of seven groups: (1) two 2-min total coronary occlusions (preconditioning, IPC) interspersed with 5 min of normal perfusion; (2) two 2-min occlusions interspersed with 5 min of perfusion while perfusing with GLB (IPC+GLB); (3) SPC (3.5%) for two 2-min periods; (4) SPC+GLB for two 2-min periods; (5) no treatment before ischemia (control [CON]); (6) CON+GLB; and (7) no ischemia (time control). Six minutes after ending IPC or SPC, hearts of ischemic groups were subjected to 30 min of global ischemia and 75 min of reperfusion. Left-ventricular pressure, coronary flow, and effluent NO concentration ([NO]) were measured. Flow and NO responses to bradykinin, and nitroprusside were tested 20-30 min before ischemia or drug treatment and 30-40 min after reperfusion. RESULTS: After ischemia, compared with before (percentage change), left-ventricular pressure and coronary flow, respectively, recovered to a greater extent (P<0.05) after IPC (42%, 77%), and treatment with SPC (45%, 76%) than after CON (30%, 65%), IPC+GLB (24%, 64%), SPC+GLB (20%, 65%), and CON+GLB (28%, 64%). Bradykinin and nitroprusside increased [NO] by 30+/-5 (means +/- SEM) and 29+/-4 nM, respectively, averaged for all groups before ischemia. [NO] increased by 26+/-6 and 27+/-7 nM, respectively, in SPC and IPC groups after ischemia, compared with an average [NO] increase of 8+/-5 nM (P<0.01) after ischemia in CON and each of the three GLB groups. Flow increases to bradykinin and nitroprusside were also greater after SPC and IPC. CONCLUSIONS: Preconditioning with sevoflurane, like IPC, improves not only postischemic contractility, but also basal flow, bradykinin and nitroprusside-induced increases in flow, and effluent [NO] in isolated hearts. The protective effects of both SPC and IPC are reversed by K(ATP) channel antagonism.

Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts.

OBJECTIVE: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. METHODS: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs-Ringer's solution equilibrated with 97% O2 and 3% CO2 at 37 degrees C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for L-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity > or = 1 nmol/l approximately 3 pA) and a selective gas permeable membrane. RESULTS: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (-0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/degree C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0 +/- 1 and 100 +/- 17 for 1 mumol/l serotonin, 7.5 +/- 1 and 148 +/- 18 for 100 nmol/l bradykinin, and 7.8 +/- 1 and 173 +/- 28 for 100 mumol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in L-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-L-arginine methyl ester (L-NAME) reduced CF by 2.6 +/- 1 ml/g/min and NO release by 25 +/- 8 pmol/g/min. An increase of CF of 8.0 +/- 0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30 +/- 4 nmol/l; L-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10 +/- 5 nmol/l. CONCLUSIONS: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by L-NAME.

Volatile anesthetics do not alter bradykinin-induced release of nitric oxide or L-citrulline in crystalloid perfused guinea pig hearts.

BACKGROUND: Nitric oxide (NO) and L-citrulline (L-cit) are released by endothelial NO synthase (eNOS) to induce vasodilation via guanylyl cyclase and cyclic guanosine monophosphate (cGMP). Volatile anesthetics directly reduce vascular muscle tone, but their effects on the eNOS cGMP pathway is controversial. The aim of this study was to examine the effects of anesthetics on bradykinin-induced increases in flow, NO, and L-cit in isolated hearts. METHODS: Guinea pig hearts were isolated, perfused at 55 mmHg with a crystalloid or erythrocyte perfusate at 37 degrees C, and heart rate, left ventricular pressure, coronary flow (CF), effluent pH, and oxygen tension were monitored. Effluent [NO] was measured by a Clark-type electrode (sensitivity > or = 1 nM = 3 pA) with a selectively permeable membrane. Effluent [L-cit] was measured by chromatography. Before, during, and after exposure to halothane, isoflurane, or sevoflurane, hearts were infused with as much as 100 nM bradykinin to induce increases in CF and effluent release of NO and L-cit. RESULTS: In crystalloid-perfused hearts, 10 nm bradykinin produced maximal concentration-dependent increases in CF (87+/-2%), [NO] (24+/-4 nM), NO release (128+/-18 pmol x g(-1) x min(-1)), and [L-cit] (58+/-8 nM). Isoflurane slightly increased CF but not NO. Anesthetics did not alter the bradykinin-induced CF, NO slope relationship, or change [L-cit]. In erythrocyte-perfused hearts, isoflurane also did not alter the bradykinin-induced increase in CF and decrease in percentage of oxygen extracted. CONCLUSIONS: This is the first study to simultaneously measure CF with bradykinin-induced changes in [NO] and [L-cit] in the presence of halothane, isoflurane, and sevoflurane in intact hearts. The study shows for the first time that volatile anesthetics do not alter the CF to NO relationship and suggests that NO production, NO release, and NO vasodilatory effects mediated by the eNOS cGMP pathway are not significantly affected by anesthetics in crystalloid or erythrocyte-perfused guinea pig hearts.

Reversal of hypothermia-induced action potential lengthening by the KATP channel agonist bimakalim in isolated guinea pig ventricular muscle.

1. ATP-sensitive potassium (KATP) channel openers shorten cardiac ventricular muscle action potential duration (APD), reduce resting and developed contractile force, and have been shown to provide cardioprotection when given before, during, and after either short-term ischemia or long-term hypothermia. The authors' aim was to determine the concentration-dependent effect of the potent KATP channel opener bimakalim on transmembrane action potential changes induced by mild (27 degrees C) and moderate (20 degrees C) hypothermia in isolated guinea pig ventricular muscle. 2. Conventional microelectrode techniques were used to record action potentials (APs) in single myocytes during normothermia (37 degrees C) and hypothermia in the presence and absence of 0.1 to 30 mumol.l-1 bimakalim. 3. Hypothermia alone increased APD and depolarized the diastolic membrane potential (DMP): APD90 = 141.7 +/- 7.0 msec and DMP -86.2 +/- 1.4 mV (n = 6) at 37 degrees C versus 235.7 +/- 7.8 msec and -75.6 +/- 1.0 mV at 20 degrees C (n = 7). At 37 degrees C, bimakalim (0.1-10 mumol.l-1) shortened APD in a concentration-dependent fashion. 4. APD90 was markedly reduced from 141.7 +/- 7.0 msec without bimakalim to 9.5 +/- 2.6 msec with 10 mumol.l-1 bimakalim (n = 6); this effect was blocked by glibenclamide. DMP was hyperpolarized by bimakalim. More bimakalim was required to shorten APs during mild and moderate hypothermia. The 50% effective concentration (EC50) of bimakalim required to maximally shorten APD90 was 0.96 +/- 0.10 mumol.l-1 at 37 degrees C; this increased to 3.96 +/- 0.24 mumol.l-1 at 27 degrees C, and to 12.34 +/- 0.72 mumol.l-1 at 20 degrees C. Relative to hypothermia-induced depolarization, bimakalim hyperpolarized DMP toward drug-free values obtained at 37 degrees C. 5. These results indicate that hypothermia shifts the bimakalim concentration APD90 response curve to the right such that 13 times more bimakalim is required at 20 degrees C shorten APD by the same amount as at 37 degrees C. Bimakalim also reverses hypothermia-induced AP lengthening and tends to reverse the hypothermia-induced decrease in DMP. 6. These findings aid in our understanding of the cardioprotective effects of KATP channel openers during hypothermia.

Improved contractility and coronary flow in isolated hearts after 1-day hypothermic preservation with isoflurane is not dependent on K(ATP) channel activation.

BACKGROUND: Isoflurane protects against reperfusion injury in isolated hearts when given before, during, and initially after hypoxia or ischemia and aids in preconditioning hearts if given before ischemia. The aims of the current study were to determine if isoflurane is cardioprotective during 1-day, severe hypothermic perfusion and if a mechanism of protection is K(ATP) channel activation. METHODS: Guinea pig hearts (n = 60) were isolated, perfused with Kreb's solution initially at 37 degrees C, and assigned to either a nontreated warm, time control group or one of five cold-treated groups: drug-free cold control, 1.3% isoflurane, 1.3% isoflurane plus glibenclamide (4 microM), 2.6% isoflurane, or 2.6% isoflurane plus glibenclamide. Isoflurane and glibenclamide were given 20 min before hypothermia, during low-flow hypothermia (3.8 degrees C) for 22 h, and for 30 min after rewarming to 37 degrees C. Heart rate, left ventricular pressure, %O2 extraction, and coronary flow were measured continuously, and responses to epinephrine, adenosine, 5-hydroxytryptamine, and nitroprusside were examined before and after hypothermia. RESULTS: Each group had similar initial left ventricular pressures, coronary flows, and responses to adenosine, 5-hydroxytryptamine, and nitroprusside. Before hypothermia, isoflurane with or without glibenclamide increased coronary flow while decreasing left ventricular pressure and %O2 extraction. After hypothermia, left ventricular pressure and coronary flow were reduced in all cold groups but least reduced in isoflurane-treated groups. During normothermic perfusion after isoflurane and glibenclamide, left ventricular pressure, coronary flow, %O2 extraction, and flow responses to adenosine, 5-hydroxytryptamine, and nitroprusside were similarly improved in isoflurane and isoflurane-plus-glibenclamide groups over the cold control group but not to levels observed in the warm-time control group. CONCLUSION: Isoflurane, like halothane, given before, during, and initially after hypothermia markedly improved but did not restore cardiac perfusion and function. Protective effects of isoflurane were not concentration dependent and not inhibited by the K(ATP) channel blocker glibenclamide. Volatile anesthetics have novel cardioprotective effects when given during long-term severe hypothermia.

Reversal of endothelin-induced vasoconstriction by endothelium-dependent and -independent vasodilators in isolated hearts and vascular rings.

Endothelin (ET-1) is a potent endogenous vasoconstrictor. Several factors increase ET-1 release in vitro and ET-1 levels increase in vivo in situations that damage blood vessels. The aim of this study was to test the activity of several differently acting vasodilator drugs on reversing or attenuating the vasoconstrictor effects of exogenously administered ET-1 in isolated guinea-pig hearts, in isolated rings with intact endothelium from canine middle cerebral and basilar arteries, and from guinea-pig aortas. Vasodilator drugs tested up to maximal concentrations were adenosine (ADE), nitroprusside (NP), acetylcholine (ACH), nifedipine (NIF), and butanedione monoxime (BDM), an excitation-uncoupling agent. Variables measured in isolated hearts included coronary flow, percentage oxygen extraction (% O2E), left ventricular pressure (LVP), and myocardial oxygen consumption. It was found that ADE, NP, ACH, and BDM each attenuated the 60% decrease in coronary flow and 20% increase in % O2E elicited by 0.5 nM ET-1 in isolated hearts, but only BDM restored coronary flow, whereas BDM and ADE both restored % O2E. In isolated rings constricted with 20 nM ET-1, BDM restored tone equivalent to that by papaverine, whereas NP and NIF only attenuated the vasoconstriction elicited by ET-1. Ring experiments also demonstrated that the vasodilatory effect of BDM was independent of nitric oxide-dependent pathways and that BDM attenuated vasoconstriction resulting from increased bath KCl. The study suggests that drugs affecting intracellular Ca2+ with a mechanism of action downstream from cell-membrane receptors or intracellular messengers may be more effective for reversing the constrictor effect of ET-1. NP, however, would be a better clinical choice for reversing ET-1-induced vasoconstriction.